Reduced Proteolysis of Rabbit Growth Hormone (GH) Receptor Substituted with Mouse GH Receptor Cleavage Site

Xiangdong Wang, Kai He, Mary Gerhart, Jing Jiang, Raymond J. Paxton, Ram K. Menon, Roy A. Black, Gerhard Baumann and Stuart J. Frank

Department of Medicine (X.W., K.H., J.J., S.J.F.), Division of Endocrinology, Diabetes, and Metabolism, and Department of Cell Biology, University of Alabama at Birmingham, Birmingham, Alabama 35294-0012; Amgen Incorporated (M.G., R.J.P., R.A.B.), Seattle, Washington 98101; Department of Pediatrics (R.K.M.), University of Michigan, Ann Arbor, Michigan 48109; Center for Endocrinology, Metabolism (G.B.), and Molecular Medicine, Department of Medicine, Northwestern University Medical School, and the Veterans Administration Chicago Health System, Lakeside Division, Chicago, Illinois 60611; and Endocrinology Section (S.J.F.), Medical Service, Veterans Affairs Medical Center, Birmingham, Alabama 35233

Address all correspondence and requests for reprints to: Stuart J. Frank, University of Alabama at Birmingham, 1530 3rd Avenue South, BDB 861, Birmingham, Alabama 35294-0012. E-mail: sjfrank{at}uab.edu.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
GH binding protein (GHBP) is a circulating form of the GH receptor (GHR) extracellular domain, which derives by alternative splicing of the GHR gene (in mice and rats) and by metalloprotease-mediated GHR proteolysis with shedding of the extracellular domain as GHBP (in rabbits, humans, and other species). Inducible proteolysis of either mouse (m) or rabbit (rb) GHR is detected in cell culture in response to phorbol ester and other stimuli, yielding a cell-associated GHR remnant (comprised of the cytoplasmic and transmembrane domains and a small portion of the proximal extracellular domain) and down-regulating GH signaling. In this report, we map the mGHR cleavage site by adenoviral overexpression of a membrane-anchored mGHR mutant lacking its cytoplasmic domain and purification and N-terminal sequencing of the phorbol 12-myristate 13-acetate-induced remnant protein. The sequence obtained was LEACEEDI, which matches the mGHR extracellular domain stem region sequence L265EACEEDI272, indicating that mGHR cleavage occurs in the extracellular domain nine residues outside of the transmembrane domain, in the same region (but at different residues) as the rbGHR cleavage site we recently mapped. We studied the effects on receptor proteolysis and GHBP shedding of replacing rbGHR cleavage site residues with those corresponding to the mGHR cleavage site. We analyzed five separate rodentized rbGHR mutants incorporating mGHR amino acids either at or surrounding the cleavage site. Each mutant was normally processed, displayed at the cell surface, and responded to GH stimulation by undergoing tyrosine phosphorylation. Only the mutants replaced with mGHR cleavage site residues, rather than surrounding residues, exhibited deficient inducible proteolysis and GHBP shedding. These findings suggested that the GHR cleavage sites in the two species differ in their susceptibility to cleavage. This difference may underlie interspecies variation in utilization of proteolysis to generate GHBP.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE GH RECEPTOR (GHR) is a type I cell surface glycoprotein that is critical in allowing GH to exert its somatogenic and metabolic effects in many species [Ref. 1 (www.academicpress.com/cytokinereference, 24-h free access)]. GH binding to the GHR in its various target tissues causes activation of the receptor-associated cytoplasmic tyrosine kinase, Janus kinase 2 (JAK2), and consequent engagement of the signal transducer and activator of transcription 5, ERK, and phosphatidylinositol-3 kinase signaling pathways (1, 2, 3). Modulation of activation of these pathways is critical for proper physiological regulation of GH action.

Our previous studies suggested that one mechanism of modulation is a metalloprotease-mediated proteolysis of the GHR, which can be mediated by the transmembrane metalloprotease, TACE (TNF{alpha}-converting enzyme; ADAM-17) (4, 5, 6, 7, 8). This proteolysis reduces surface receptor content and concomitantly generates a soluble extracellular domain (ECD) (referred to as the GH binding protein or GHBP) and a cell-associated receptor remnant that includes the transmembrane and cytoplasmic domains. This remnant could, in principle, impact GH signaling by associating with intracellular signaling molecules, but its physiological significance remains as yet unknown. While GHBP generation in rabbits (and humans) is believed to result from receptor proteolysis and ECD shedding, mice (and rats) instead utilize alternative mRNA splicing as a mechanism to generate their GHBP (9, 10, 11, 12). In that case, GHBP consists of the GHR ECD fused to a short hydrophilic peptide encoded by an exon not found in the mature GHR transcript. Interestingly, although shedding has not been demonstrated to be a mechanism of rodent GHBP generation in vivo, metalloproteolytic desensitization of GH signaling can be observed in cells expressing either rabbit (rb) or mouse (m) GHRs (6, 7).

The observation that GHR proteolysis may be differentially used among species as a GHBP-generating vs. signal modulation mechanism has encouraged us to carefully compare the determinants of receptor proteolysis in the rb- and mGHRs. We recently reported the cleavage site in the ECD of the rbGHR (7). By adenovirally overexpressing a membrane-anchored rbGHR mutant lacking its cytoplasmic domain, we were able to purify and N-terminally sequence the phorbol 12-myristate 13-acetate (PMA)-induced remnant peptide. This analysis suggests cleavage occurs eight residues from the membrane in the proximal ECD stem region of the receptor. The ECD stems of rbGHR and mGHR are highly similar, but differ most in the region corresponding to the mapped rbGHR cleavage site (10, 13). In this report, we map the cleavage site of mGHR and compare it to that previously mapped for the rbGHR.1 Further, we study the effects of replacing rbGHR cleavage site residues with those mapped for the mGHR. Our analysis suggests that the cleavage sites in the receptors of the two species differ in their intrinsic cleavability (i.e. inherent sensitivity to cleavage) and that this difference may underlie interspecies variation in utilization of proteolysis to generate GHBP.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Adenoviral Expression of C-Terminal Epitope-Tagged mGHR Cytoplasmic Domain Mutant and Mapping of the mGHR Cleavage Site
We have observed metalloprotease-mediated proteolysis of both the mGHR (in 3T3-F442A cells) and rbGHR (expressed in a variety of cells), although proteolytic GHBP shedding is much less for mGHR than rbGHR (5, 6, 7). To compare the mGHR and rbGHR structural determinants for proteolytic cleavage, we sought to map the mGHR cleavage site. We used an adenoviral overexpression system in human embryonic kidney (HEK)-293 cells similar to that which we described previously for mapping of the rbGHR cleavage site (7, 14). The pAdlox system was employed to prepare infectious adenoviral particles encoding a C-terminally Myc-His-tagged mGHR that includes only residues 1–301 (mGHR1-301-Myc-His) (1). This truncation mutant contains the mGHR ECD and transmembrane domain (TMD) and only the first four cytoplasmic domain residues followed by C-terminal Myc and His tags. It is analogous to both a naturally occurring GHR splice variant (15, 16) and our previously characterized rbGHR1-274-Myc-His (7), which was used to determine the rbGHR cleavage site.

We first tested the expression and proteolytic cleavage of mGHR1-301-Myc-His in adenovirally infected HEK-293 cells (Fig. 1Go). Infected cells were serum starved and then treated with either PMA or vehicle control for 45 min, harvested, and detergent solubilized. Extracted proteins were resolved by SDS-PAGE and immunoblotted with an anti-Myc monoclonal antibody. As expected based on our previous findings (7, 17, 18), mGHR1-301-Myc-His was detected as a broad, indistinct band migrating at 65–90 kDa, consistent with its being a glycoprotein (Fig. 1AGo, bracket). Treatment with PMA caused the enhanced appearance of an additional anti-Myc-reactive band of Mr roughly 16 kDa (Fig. 1AGo, lane 2 vs. 1, arrow), consistent with being the GHR remnant we previously described as appearing in response to PMA for both mGHR and rbGHR forms (6, 7, 18). To determine whether this lower band might indeed be the remnant, we examined the effect of the metalloprotease inhibitor, Immunex compound 3 (IC3), on its generation. Indeed, short-term incubation with IC3 markedly inhibited PMA-induced remnant appearance (lane 3 vs. 2), suggesting metalloprotease dependence for this cleavage. We also assessed whether there was release of GHBP into the medium of the cells that expressed adenovirally produced mGHR1-301-Myc-His. Supernatants of cells incubated in the presence of PMA or vehicle for 45 min were tested for GHBP by a [125I]GH binding assay (Fig. 1BGo). GHBP was detected in both instances, but PMA substantially increased its production. Inclusion of IC3 markedly inhibited both basal and PMA-induced GHBP production. These findings strongly suggest that basal and inducible proteolysis of mGHR1-301-Myc-His occurred in this adenoviral system in a fashion similar to our previous observations for the rbGHR in this same system (7).



View larger version (13K):
[in this window]
[in a new window]
 
Fig. 1. Inducible Metalloprotease-Mediated Receptor Proteolysis and GHBP Shedding of Adenovirally Expressed mGHR1-301-Myc-His

A, Receptor proteolysis. HEK-293 cells were adenovirally infected with pAdlox-mGHR1-301-Myc-His, as in Materials and Methods. Serum-starved cells (one 90% confluent well of a six-well plate per sample) were exposed in samples 1–4 to PMA (+) or the dimethylsulfoxide vehicle (-) for 45 min in the presence (+) or absence (-, dimethylsulfoxide vehicle) of IC3 (50 µM). At the end of the incubations, one of six of the detergent cell extracts were resolved by SDS-PAGE and immunoblotted with anti-Myc antibodies. The positions of the GHR and the remnant are indicated, as are the migrations of prestained molecular mass markers. The data shown are representative of two such experiments. B, GHBP shedding. Supernatants from samples such as in A were harvested before cell lysis. GHBP content was measured in the supernatants, as in Materials and Methods, and is graphically expressed as percent of radiolabeled GH bound per 50 µl of medium. Note that IC3 inhibits both PMA-induced and basal GHBP shedding for rbGHR1-301-Myc-His expressed in this system.

 
We next used the adenoviral system to produce mGHR1-301-Myc-His remnant of sufficient quantity to obtain N-terminal sequence information. The strategy employed for scaling up and purification is detailed in Materials and Methods. In brief, large quantities of infected HEK-293 cells were treated with PMA under serum-starved conditions at 37 C for 45 min, after which they were harvested and detergent solubilized. The C-terminally His-tagged remnant was enriched from the detergent cell extract by metal affinity chromatography and imidazole elution, resolved by SDS-PAGE, and subjected to Western transfer. The band of interest was excised from the nitrocellulose filter and submitted for N-terminal sequencing by Edman degradation. The amino acid sequence obtained was LEACEEDI, which matches the mGHR ECD stem region sequence L265EACEEDI272 (10), verifying that we had purified and sequenced the protein of interest (Fig. 2AGo). As diagrammed in Fig. 2BGo, these data suggest that cleavage of the mGHR (designated m) that leads to remnant generation occurs between residues I264 and L265, with L265 being in the ECD nine residues outside of the first TMD residue.



View larger version (22K):
[in this window]
[in a new window]
 
Fig. 2. Purification of GHR Remnant and Sequencing of its N Terminus

A, Sequence data. Large-scale adenoviral expression of mGHR1-301-Myc-His and the PMA-induced generation of and purification of its remnant protein were performed as in Materials and Methods and the text. Edman degradation sequencing yielded the sequence LEACEEDI as the N-terminal sequence of the purified remnant (~2.6 pmol was recovered, based on the sequencing results). B, Comparison of mouse (m), rat (r), rabbit (rb), and human (h) GHR amino acid sequence in the juxtamembraneous ECD, TMD (boxed), and juxtamembraneous intracellular (ICD) domain regions. Arrowhead indicates the mapped cleavage sites for the mGHR and rbGHR, as in the text.

 
For comparison, the amino acid sequences of the TMD and perimembraneous ECD and intracellular domains of the mouse GHR (m), rat GHR (r), rabbit GHR (rb), and human GHR (h) are aligned in Fig. 2BGo. The cleavage sites of the mGHR and rbGHR mapped by us (this report and Ref.7 , respectively) are indicated by arrowheads. We note that the transmembrane regions of all four species are nearly identical and that the proximal ECD regions shown for each are also quite similar, except for the amino acids surrounding the cleavage sites (N263ILEA for mGHR and S237PFT for rbGHR, indicated by brackets). The corresponding rGHR and hGHR regions are also bracketed, with rGHR appearing more similar to mGHR and hGHR appearing more similar to rbGHR in those regions. Notably, the spacing of amino acids differs in this bracketed region, with the mGHR and rGHR including five residues and the rbGHR and hGHR including four residues. Given that the predominant mechanisms of GHBP production in mice and rats is believed to be alternative mRNA splicing rather than proteolysis and that rabbits and humans generate their GHBP by proteolysis, the amino acid differences in the cleavage regions of mGHR and rbGHR suggested a potential basis for the interspecies differences in proteolysis.

Generation and Characterization of rbGHR Cleavage Region Mutants Replaced with mGHR Elements
We previously studied determinants of rbGHR cleavage by performing transient expression studies in HEK-293 cells (7). In this system, we used rbGHRdel 297-406, an rbGHR with in-frame internal deletion of cytoplasmic domain residues 297–406. Our prior work has shown that rbGHRdel 297-406, which lacks the internalization and UbE motif (19, 20), is highly expressed at the cell surface, normally couples to GH-induced JAK2 activation, and undergoes inducible metalloprotease-mediated proteolysis and GHBP shedding (7, 17, 18, 21). Because it has a normal ECD and TMD, we used rbGHRdel 297-406 as our framework GHR (referred to as WT for wild-type) into which mutations were introduced into the cleavage region to test their effects on GHR proteolysis and GHBP shedding.

WT rbGHR and the mutants to be studied are diagrammed in Fig. 3Go. rbGHR-{Delta}237–239 and rbGHR-237–239AAA are two previously characterized mutants that harbor either deletion of residues S237PF or replacement of these residues with alanines, respectively. rbGHR-{Delta}237–239 does not undergo proteolysis or GHBP shedding, whereas rbGHR-237–239AAA is inducibly cleaved and sheds GHBP normally (7). The remaining five mutants were prepared to test the effects on GHR proteolysis and GHBP shedding of replacing the rbGHR cleavage site and/or surrounding residues with corresponding regions of the mGHR. We thus refer to these mutants as "rodentized" rbGHRs. rbGHR-NILEA/SPFT has the S237PFT rabbit sequence replaced with the N263ILEA mouse sequence; thus, the two residues on either side of the rbGHR cleavage site are replaced with the two residues N-terminal and three residues C-terminal to the mGHR cleavage site. To retain the cleavage site swap, but avoid changing the number of amino acids, we created two other mutants, rbGHR-NIL/SPF and rbGHR-IL/PF. In both mutants, the particular rbGHR cleavage residues (with one residue N-terminal to it in rbGHR-NIL/SPF and only the cleavage residues in rbGHR-IL/PF) are replaced by the analogous mGHR cleavage residues we mapped above in Fig. 2Go. In the remaining two rodentized mutants, the rbGHR cleavage site residues are maintained, but the spacing of the mGHR (an extra residue in comparison to rbGHR) is adopted by either 1) replacing rbGHR 240T (the final residue in the SPFT sequence) with EA (the final two residues in the mGHR NILEA sequence) to yield rbGHR-EA/T; or 2) inserting an alanine (the final residue of the mGHR NILEA sequence) between rbGHR residues 240T (the final residue of the SPFT sequence) and 241C to yield rbGHR-A into TC.



View larger version (26K):
[in this window]
[in a new window]
 
Fig. 3. GHR Mutants to be Studied

rbGHRdel 297-406, which has an in-frame internal deletion of 110 cytoplasmic domain residues, including the internalization and UbE motifs (see text), is diagrammed. This is referred to as WT, for comparison to the other mutants, which also have deletions, alanine substitutions, or replacement of rbGHR sequences with mGHR sequences in the juxtamembraneous ECD. For the deletion ({Delta}) mutant, rbGHR-{Delta}237–239, the residues deleted are indicated by their absence and replacement with an underline. For rbGHR-237–239AAA, in which a contiguous group of three residues are replaced by alanine (AAA), the residue changes are indicated. For the new mutants that harbor elements of the mGHR sequence, the residues that replace the rbGHR residues are indicated. The positions of phenylalanine-239 (F239) (in the rbGHR sequence) and leucine-265 (L265) (in the mGHR sequence) are indicated, and the mapped cleavage sites (bold arrowheads) between residues 238 and 239 in rbGHR and between residues 264 and 265 in mGHR are indicated.

 
Each of the five rodentized rbGHR mutants harbors changes in the ECM stem region, a region not directly involved in GH binding (22). Our previous analysis of rbGHR-{Delta}237–239 and rbGHR-237–239AAA indicated that both displayed normal glycoprotein processing and both responded to GH by activating JAK2 (7). Nevertheless, we sought to verify the structural integrity of our new stem region mutants before testing their sensitivity to proteolysis. We first addressed the processing of the mutants by testing their susceptibility to deglycosylation by endoglycosidase H (endoH) (Fig. 4AGo). As with other N-glycosylated membrane proteins, sensitivity of the GHR to endoH deglycosylation indicates the presence of immature high-mannose receptor forms that have yet to traverse the Golgi apparatus, whereas the acquisition of resistance to endoH deglycosylation indicates a GHR with a mature pattern of carbohydrate modification that occurs at a post-trans-Golgi location. Thus, endoH-resistant GHRs are considered cell surface-destined mature forms of the protein present within the mix of variably glycosylated forms typically detected in cells (23, 24).



View larger version (35K):
[in this window]
[in a new window]
 
Fig. 4. EndoH Sensitivity and Surface [125I]GH Binding of WT and rbGHR Mutants

A, EndoH sensitivity. HEK-293 cells were transiently transfected with either pcDNA 3.1-driven WT (rbGHRdel 297-406), rbGHR-NILEA/SPFT, rbGHR-NIL/SPF, rbGHR-IL/PF, rbGHR-EA/T, or rbGHR-A into TC, as indicated, plus pcDNA 3.1 JAK2. Serum-starved cells (one 90% confluent 60 x 15 mm dish per sample) were harvested and detergent extracted. GHRs were immunoprecipitated with anti-GHRcyt-mAb. Precipitated proteins were divided equally and treated either with (+) or without (-) endoH and eluates were resolved by SDS-PAGE and immunoblotted with anti-GHRcyt-AL47. The positions of the endoH-resistant and endoH-sensitive forms of each receptor are indicated, as is the position of the deglycosylated form that appears in response to endoH (endoH-sensitive degly). Note that each mutant displayed the same pattern of endoH sensitivity as did the WT. The data shown are representative of three such experiments. B, [125I]GH binding. Transiently transfected HEK-293 cells expressing WT or the indicated rbGHR mutants plus pcDNA 3.1 JAK2 (duplicate six-well wells per condition) were serum starved and incubated with [125I]hGH in the absence or presence of unlabeled hGH (2 µg/ml) at 25 C for 1 h. Cells were washed and solubilized, as outlined in Materials and Methods. Bound radioactivity was measured by {gamma}-counting. Replicate samples of serum-starved cells were detergent extracted, and cellular proteins were resolved by SDS-PAGE and immunoblotted with anti-GHRcyt-AL47. Specific binding was normalized by densitometrically determined GHR abundance for each sample and displayed relative to the same ratio for WT (considered 100%) within each experiment. Data are plotted as mean ± SE for n = 2. P > 0.2 for the comparison of each mutant with WT.

 
The mutant rbGHRs were each transiently expressed in HEK-293 cells. Detergent-extracted proteins were immunoprecipitated with our monoclonal antibody that recognizes the receptor cytoplasmic domain (anti-GHRcyt-Mab) (21), and immunocomplexes were subjected to treatment with endoH or its vehicle control before elution, resolution by SDS-PAGE, and immunoblotting with an anti-GHR cytoplasmic domain serum (anti-GHRcyt-AL47) (Fig. 4AGo). As expected, each immunoprecipitated receptor not treated with endoH was detected as a mixture of forms including a broad set of bands migrating at Mr of approximately 85–100 kDa and a sharper band at approximately 75 kDa (Fig. 4AGo, lanes 1, 3, 5, 7, 9, and 11). As we previously observed for WT rbGHR, rbGHR-{Delta}237–239, and rbGHR-237–239AAA (7), endoH treatment of each mutant selectively converted the sharper band to a faster migrating form, identifying it as the immature or precursor form (Fig. 4AGo, lanes 2, 4, 6, 8, 10, and 12). The lack of effect on the 85- to 100-kDa broad bands by endoH signified that each mutant also exists in the endoH-resistant (mature) form. This pattern for the mutants was similar to that found for WT rbGHR, suggesting that each mutant was processed normally, despite the ECD mutations.

We further characterized the mutants by comparing the surface GH binding of each with that of WT rbGHR when expressed in HEK-293 cells (Fig. 4BGo). After transient transfection with the GHR forms, the [125I]hGH binding capacity of each was determined, as detailed in Materials and Methods. When normalized for the expression of each receptor (by anti-GHRcyt-AL47 immunoblotting), there were no substantial differences among the mutants and WT rbGHR in the surface-radiolabeled GH binding. This complements the endoH deglycosylation data to indicate that surface routing of these mutants is apparently intact and that the GH binding sites are not disrupted.

We next tested the ability of each rbGHR (WT or new mutants) to allow GH-induced signaling by examining the effect of GH on GHR and JAK2 tyrosine phosphorylation (Fig. 5Go). WT rbGHR or each of the rodentized rbGHR mutants was cotransfected with JAK2 into HEK-293 cells. Serum-starved transfected cells were treated with GH or vehicle for 10 min before harvesting, detergent solubilization, SDS-PAGE, and sequential immunoblotting with antiphosphotyrosine (anti-pTyr, upper panel) and anti-JAK2 (lower panel) antibodies. As we have found previously (7), transfection with vector only revealed neither GHR nor JAK2 tyrosine phosphorylation, given the absence of immunodetectable GHR in these cells (data not shown). In concert with our previous observation for rbGHRdel 297-406 (17), basal JAK2 tyrosine phosphorylation was observed upon coexpression of JAK2 with either WT rbGHR or mutants. Some basal (GH-independent) GHR tyrosine phosphorylation was also observed, the degree of which generally correlated with the relative level of JAK2 expressed (compare lanes 1, 3, 5, 7, 9, 11, and 13, upper vs. lower panels). Notably, in each case, the addition of GH caused increased GHR tyrosine phosphorylation, suggesting that, consistent with our previous data for rbGHR-{Delta}237–239 and rbGHR-237–239AAA (7), each of the rodentized rbGHR mutants productively bound GH to initiate signal transduction. In each case, a similar pattern of multiple bands was noted, perhaps consistent with the presence of receptor species with variable glycosylation and/or varying levels of tyrosine phosphorylation.



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 5. WT and Mutants Are Each Able to Respond to GH with JAK2 and GHR Tyrosine Phosphorylation

HEK-293 cells were transiently transfected with pcDNA 3.1-driven WT (rbGHRdel 297-406) or mutants, as indicated, plus pcDNA 3.1 JAK2. Serum-starved cells (one 90% confluent 60 x 15 mm dish per sample) were treated with (+) or without (-) GH for 10 min. Detergent cell extracts were resolved by SDS-PAGE and sequentially immunoblotted with anti-pTyr (upper panel) or anti-JAK2AL33 (lower panel). The positions of tyrosine-phosphorylated JAK2, total JAK2, and tyrosine-phosphorylated GHR are indicated. The data shown are representative of three such experiments.

 
Proteolytic Susceptibility of rbGHR Cleavage Region Mutants Replaced with mGHR Elements
Our previous work indicated that deletion of the S237PF rbGHR residues (rbGHR-{Delta}237–239) eliminated receptor proteolysis and GHBP shedding, but that replacement of these residues with alanine (rbGHR-237–239AAA) allowed normal proteolytic processing (7). To determine the effects of replacement of rbGHR cleavage site region residues with mGHR residues, we tested the mutants described in Figs. 3–5GoGoGo for their susceptibility to proteolysis. In Fig. 6AGo, the results of three representative experiments are shown. In each, HEK-293 cells were transiently transfected with the WT or mutant rbGHRs, and serum-starved cells were treated with or without PMA for 45 min at 37 C. Extracted proteins were resolved by SDS-PAGE and immunoblotted with anti-GHRcyt-AL47, which detects both the GHR (bracket) and the cytoplasmic domain remnant (arrowhead). In the experiment shown in lanes 1–8, comparison of expression of vector only (lanes 1 and 2), WT rbGHR (lanes 3 and 4), rbGHR-{Delta}237–239 (lanes 5 and 6), and rbGHR-237–239AAA (lanes 7 and 8) is made and shows that, consistent with our previous data, basal and PMA-inducible remnant appeared for the WT and rbGHR-237–239AAA, but not for rbGHR-{Delta}237–239.



View larger version (35K):
[in this window]
[in a new window]
 
Fig. 6. Proteolysis and Shedding of the GHR Mutants

A and B, Receptor proteolysis. A, In three representative independent experiments, HEK-293 cells were transiently transfected with either vector (pcDNA 3.1) only (lanes 1, 2), or pcDNA 3.1-driven WT (rbGHRdel 297-406) or mutants, as indicated (lanes 3–24), plus pcDNA 3.1 JAK2, as in Materials and Methods. Serum-starved cells (one 90% confluent well of a six-well plate per sample) were exposed to PMA (+) or the dimethylsulfoxide vehicle (-) for 45 min. Detergent cell extracts were resolved by SDS-PAGE and immunoblotted with anti-GHRcyt-AL47. The positions of the GHR (brackets), its remnant (arrowheads), and the prestained molecular mass markers are shown. Note the lack of remnant generated from rbGHR-{Delta}237–239 and the diminished remnant generation from rbGHR-NILEA/SPFT, rbGHR-NIL/SPF, and rbGHR-IL/PF. B, Densitometric analysis of experiments such as those shown in panel A. As described in Materials and Methods, abundance of remnant normalized for abundance of receptor in each sample are compared within each experiment to the same ratio for WT. Data are plotted as mean ± SE. The number of replicates evaluated for each receptor were: WT, n = 8; rbGHR-{Delta}237–239, n = 3; rbGHR-237–239AAA, n = 3; rbGHR-NILEA/SPFT, n = 8; rbGHR-NIL/SPF, n = 5; rbGHR-IL/PF, n = 4; rbGHR-EA/T, n = 3; rbGHR-A into TC, n = 3. P < 0.001 for the PMA-stimulated samples of rbGHR-NILEA/SPFT, rbGHR-NIL/SPF, and rbGHR-IL/PF, each in comparison to WT. C, GHBP shedding. For WT, rbGHR-NILEA/SPFT, rbGHR-EA/T, and rbGHR-A into TC, supernatants (0.4 ml) from PMA-stimulated samples such as in panel A were harvested before cell lysis. GHBP content was measured in the supernatants, as in Materials and Methods. Within each of two independent experiments, the amount of shed GHBP was normalized for the abundance of the transfected receptor by densitometry (as in Materials and Methods). Data are graphically expressed for each mutant as the normalized GHBP shed relative to that shed by WT within each experiment and are plotted as mean ± SE (n = 2). The differences shown are significant (P < 0.04).

 
In lanes 9–16 of Fig. 6AGo, the same analysis is shown for WT rbGHR (lanes 9 and 10) compared with rbGHR-NILEA/SPFT (lanes 11 and 12), rbGHR-EA/T (lanes 13 and 14), and rbGHR-A into TC (lanes 15 and 16). Again, ample remnant was generated from WT rbGHR. In contrast, rbGHR-NILEA/SPFT, which harbors the mGHR, rather than rbGHR, cleavage region, yielded only low levels of basal or PMA-induced remnant. Notably, both the rbGHR-EA/T and rbGHR-A into TC mutants exhibited robust basal and inducible remnant generation. These mutants, unlike rbGHR-NILEA/SPFT, maintain the P238F rbGHR cleavage site, but have changes just C-terminal to that site and introduce the mGHR spacing (five residues rather than four in rbGHR) that also was introduced with the rbGHR-NILEA/SPFT mutant. These results suggest that replacement of the rbGHR cleavage residues with the mGHR cleavage residues, rather than the extra residue spacing, lessened the rbGHR cleavability. To further probe the importance of the mGHR cleavage residues in the context of the rbGHR, we compared rbGHR-NILEA/SPFT with the rbGHR-NIL/SPF and rbGHR-IL/PF mutants (Fig. 6AGo, lanes 17–24). Again, rbGHR-NILEA/SPFT was markedly deficient in remnant abundance when compared with WT rbGHR (lanes 19 and 20 vs. 17 and 18). Interestingly, both rbGHR-NIL/SPF and rbGHR-IL/PF were similarly deficient as rbGHR-NILEA/SPFT, strongly bolstering the conclusions drawn with rbGHR-NILEA/SPFT that replacement of the rbGHR cleavage site with the two residues that define the mGHR cleavage site markedly impairs rbGHR proteolysis.

We quantified data derived from multiple experiments, such as those shown in Fig. 6AGo, by estimating densitometrically the abundance of the remnant detected by immunoblotting normalized for the abundance of the receptor in the same samples. The densitometric analysis is presented in Fig. 6BGo, in which the relative remnant abundance for each condition is expressed as a percentage of that detected for PMA-stimulated cells harboring WT rbGHR within the same experiment and from the same immunoblot. This analysis confirmed the noncleavability of rbGHR-{Delta}237–239, as expected, and demonstrated the substantially reduced proteolysis of the rbGHR-NILEA/SPFT, rbGHR-NIL/SPF, and rbGHR-IL/PF mutants when compared with WT rbGHR and to the rbGHR-237–239AAA, rbGHR-EA/T, and rbGHR-A into TC mutants. This differential sensitivity to proteolysis among the mutants was also demonstrated by measurement of shed GHBP into the supernatants of HEK-293 cells. As seen in Fig. 6CGo, rbGHR-NILEA/SPFT shed substantially less GHBP than did either rbGHR-EA/T or rbGHR-A into TC. These shedding data support the findings obtained by immunoblotting of remnant as a reflection of receptor proteolysis.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The GHBP is a high-affinity binding protein for GH found in the circulation of many species. It is equivalent to a circulating version of the ECD of the GHR and carries approximately half of the circulating GH in the human (12, 25). The roles of GHBP in GH action are likely complex as it has been shown, depending on the experimental situation, to have several effects, including stabilization of GH bioavailability, sequestration of GH from cell surface GHR, or formation of inactive GH-GHBP-GHR complexes (26, 27, 28, 29). The net effect of GHBP in GH physiology remains uncertain (12).

It is clear that two different mechanisms exist for generation of GHBP and that their utilization is species dependent. In rats and mice, alternative splicing of the GHR mRNA results in a secreted form (the GHBP) that includes the ECD; a study using antibodies specific to an epitope present only in the spliced form suggested that this secreted form accounts for the vast majority, if not all, of the circulating GHBP in the rat (9, 10, 11). No such spliced mRNA has been found in humans, and it is believed that GHBP in humans, rabbits, and several other species is derived by proteolysis of GHR (12). This process yields the soluble receptor ECD (the shed GHBP) and an obligatory byproduct, the so-called GHR "remnant," a cytoplasmic domain-containing cell-associated fragment of the receptor that remains after the ECD is shed (4, 6, 7, 18). Interestingly, we have observed metalloproteolysis of the endogenous mGHR in 3T3-F442A cells and even a small amount of PMA-inducible GHBP shedding from these cells (6). Nevertheless, in our studies the amount of mGHBP shed from 3T3-F442A cells is lower by almost 2 orders of magnitude than rbGHBP shed from several different cell types that express the rbGHR by transfection.

Others have also examined the effects of expression of the full-length or cytoplasmic domain truncation mutant rbGHR and rGHR in tissue culture cells (30, 31). In those studies, GHBP was detected in the supernatants of the rbGHR-expressing cells, but none was released from the rGHR-expressing cells. It is notable that only constitutive proteolytic shedding was measured under the conditions used in those studies, as no metalloprotease inducer was employed. In addition, no assessment of proteolysis was pursued. In contrast, our studies with 3T3-F442A cells suggest that metalloprotease-mediated GHR proteolysis can be induced by stimuli that include PMA, platelet-derived growth factor, and serum (6).

In principle, the difference in proteolytic GHBP shedding between species could be explained by at least two possibilities. One possibility is a difference in the expression of the enzyme(s) responsible for this proteolytic activity and/or another associated or facilitatory molecule. We previously identified the transmembrane metalloprotease TACE (ADAM-17) as a GHBP sheddase (5) by demonstrating that reconstitution of TACE -/- fibroblasts with the rbGHR and murine TACE allowed inducible GHBP shedding. (Reconstitution with the rbGHR alone, however, failed to allow GHBP shedding.) In this experiment, mouse cells were used for reconstitution, suggesting that proteins that allow such rbGHR processing are not absent in rodent cells and that murine TACE could cleave the rbGHR.

An alternative possibility to explain the interspecies differences in GHR proteolysis is that the mGHR and rbGHR have dissimilar structural features such that receptor proteolysis is favored in one over the other species. In the current study, we have approached this issue of cleavage susceptibility using our previously defined systems for adenoviral overexpression and transient reconstitution of HEK-293 cells with an rbGHR variant into which cleaving region mutations can be introduced. We demonstrated that metalloprotease-dependent (IC3-inhibitable) remnant accumulation and GHBP shedding resulted in adenoviral overexpression of a truncated mGHR and used this mutant to map the site of cleavage in the mGHR. By comparing this site to that which we recently mapped for rbGHR (7) and examining the quite similar perimembranous extracellular regions of the mouse and rabbit receptors, we observed that the cleavage of both mGHR and rbGHR maps to that small region of the receptor extracellular stem that is least similar between the two species. Mutagenic introduction of elements of this small region of mGHR in place of the analogous rbGHR elements revealed that replacement with the mGHR cleavage site (mouse I264L for rabbit P238F in the rbGHR-IL/PF mutant) was sufficient to reduce substantially, but not eliminate, receptor proteolysis. Further, other mutants introducing nearby mouse receptor elements, but retaining the rbGHR cleavage site (rbGHR-EA/T and rbGHR-A into TC), exhibited no such decrement in proteolysis. These results suggest that the differences in the mGHR and rbGHR in the cleavage site region contribute to the interspecies difference of proteolytic GHBP shedding by affecting the intrinsic cleavability of the receptor.

Comparison of these findings to our previous observations in mapping and mutating the rbGHR cleavage site is warranted and illustrative. In our previous study (7), we found that removal of the three residues at the cleavage site (the cleavage site residues, P238F, and one amino acid N-terminal to it, in an attempt to retain helical register if a helix is indeed the structure adopted in this region), which yielded the mutant rbGHR-{Delta}237–239, resulted in complete abrogation of receptor proteolysis and GHBP shedding. Interestingly, replacement of these same residues with alanine (rbGHR-237–239AAA) completely restored (or even enhanced) both proteolysis and shedding. This and other deletion and alanine substitution mutagenesis data led us to conclude that the identity of the rbGHR cleavage site residues, per se, was likely of less importance than the maintenance of the distance of the cleavage site from the membrane. Whereas the spacing between the cleavage site and the membrane may be of importance, our current data suggest that the identity of the cleavage residues is also important. Interestingly, one interpretation of our integrated findings is that, although rbGHR cleavage tolerates certain amino acid substitutions in this region (i.e. alanines), the mGHR cleavage sequence is less tolerated and thus dampens the ability of the receptor to be proteolyzed. Indeed, this is consistent with the finding for TNF{alpha} that substitution of isoleucine for the naturally occurring alanine residue at the cleavage site substantially reduces cleavability (32, 33). Thus, it may be the adoption in the hGHR and rbGHR of a nonrodent cleavage region sequence, along with the lack of production of a suitably alternatively spliced mRNA for GHBP secretion, that accounts for the primacy of proteolysis as the GHBP-generating mechanism in humans and rabbits. Our knowledge of the structure adopted by the perimembranous GHR stem region and of the molecular mechanisms of the metalloprotease’s interaction with and cleavage of the GHR is as yet too limited to allow a more precise understanding of the basis for the cleavage sensitivity.

Finally, despite the decreased cleavage of the rodentized rbGHR, we emphasize that neither it nor the truncated mGHR was completely resistant to cleavage. This conforms to our findings in the 3T3-F442A cell, in which inducible metalloproteolysis of the mGHR was detectable and impacted upon GH signaling sensitivity (6). It may be informative in future studies to probe further the effects of reciprocal introduction of rbGHR cleavage site residues in the context of the mGHR to examine the effect of such changes on receptor cleavability and GH sensitivity. Further, in light of our findings, it may be profitable to reexamine in vivo in rodents whether physiological and pathophysiological perturbations that may impact upon GHBP levels may indeed contribute to GHBP generation in part by recruiting the shedding process.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
PMA and routine reagents were purchased from Sigma Chemical Co. (St. Louis, MO) unless otherwise noted. Restriction endonucleases were obtained from New England Biolabs (Beverly, MA). Recombinant hGH was kindly provided by Eli Lilly & Co. (Indianapolis, IN). Immunex compound 3 (IC3), supplied by Amgen, Inc. (Seattle, WA), is identical to compound 2 (34), except that the napthylalanine side chain is replaced by a tert butyl group. Talon Metal Affinity Resin, used for His-tagged protein purification, was from CLONTECH (Palo Alto, CA). [125I]hGH (specific activity 116 µCi/µg) was purchased from Perkin-Elmer Corp. (Norwalk, CT).

Cells, Cell Culture, Transfection, and Adenoviral Infection
HEK-293 cells were maintained in DMEM (low glucose) (Cellgro, Inc., Herndon, VA) supplemented with 7% fetal bovine serum (Biofluids, Rockville, MD) and 50 µg/ml gentamicin sulfate, 100U/ml penicillin, and 100 µg/ml streptomycin (all Biofluids). Transient transfection was achieved by introducing pCDNA 3.1-driven plasmids encoding GHR mutants (3 µg per transfection; see below for construction) with or without murine JAK2 (1 µg per transfection), as indicated, using Lipofectamine Plus (Invitrogen, San Diego, CA) according to the manufacturer’s instructions. Adenoviral infection of HEK-293 cells was accomplished using methods previously reported (7, 14).

Plasmid Construction
The rbGHR cDNA was a kind gift of Dr. W. Wood, Genentech, Inc. (South San Francisco, CA). Construction of the cDNA encoding rbGHRdel 297-406 has been described previously (17, 21). This mutant (referred to as WT in this report) has intact ECDs and TMDs, but lacks residues 297–406 in the cytoplasmic domain (the full-length rbGHR has 620 residues). The box 1 region in the proximal cytoplasmic domain is intact, as is the distal two thirds of the cytoplasmic domain, which contains known GHR tyrosine phosphorylation sites, but the major internalization motif is absent. Ligation of rbGHRdel 297-406 cDNA into the pcDNA 3.1 (-) eukaryotic expression vector was previously described (7).

cDNA expression plasmids encoding the rbGHR cleavage region mutants, rbGHR-{Delta}237–239 and rbGHR-237–239AAA, were previously described (7). cDNA expression plasmids encoding the rbGHR cleavage region chimera mutants, rbGHR-NILEA/SPFT, rbGHR-NIL/SPF, rbGHR-IL/PF, rbGHR-EA/T, and rbGHR-A into TC, were each constructed using the ExSite (Stratagene, La Jolla, CA) PCR-based site-directed mutagenesis method and the pCDNA3.1-rbGHRdel 297-406 as the template. The resulting mutants are diagrammed in Fig. 3Go. In each case, the mGHR sequences introduced in place of the rbGHR sequences are indicated. Sequences for the mutagenic oligonucleotides are available upon request. The entire protein coding sequence of each mutant cDNA was subjected to dideoxy DNA sequencing (UAB core facility), which verified the presence of the desired mutations and the absence of unwanted mutations.

Generation of Recombinant Adenoviruses
The methods for generating the adenovirally expressed version of mGHR1-301-Myc-His were described previously (14). Briefly, linearized pAdlox-mGHR1-301-Myc-His and {psi}5 helper virus DNA were cotransfected into CRE8 cells (35) (an HEK-293 derivative) by lipofectamine (Invitrogen). The cells were harvested after several days when cytopathic effects became apparent. After lysis by three freeze-thaw cycles, cell debris was pelleted by centrifugation and supernatant was collected. This supernatant was used for infection of HEK-293 cells. Three further rounds of infection were performed to obtain a high-titer viral stock, which was used for experimental and preparative infection.

Antibodies
The 9E10 anti-Myc monoclonal antibody and the 4G10 monoclonal anti-pTyr antibody were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). The rabbit polyclonal antiserum, anti-GHRcyt-AL47, raised against a bacterially expressed N-terminally-His-tagged fusion protein incorporating human GHR residues 271–620 [the entire cytoplasmic domain (13)], has been previously described (18). Anti-GHRcyt-mAb is a mouse monoclonal antibody directed against a bacterially expressed glutathione-S-transferase fusion protein incorporating human GHR residues 271–620 and has been previously described (21). Anti-JAK2AL33 (directed at residues 746-1129 of murine JAK2) polyclonal serum has been described (36).

Cell Stimulation, Protein Extraction, Immunoprecipitation, Deglycosylation, Electrophoresis, and Immunoblotting
Serum starvation of HEK-293 transfectants and infectants was accomplished by substitution of 0.5% (wt/vol) BSA (fraction V, Roche Clinical Laboratories, Indianapolis, IN) for serum in their respective culture media for 16–20 h before experiments. Unless otherwise noted, stimulations were performed at 37 C. Details of the hGH (500 ng/ml) and PMA (at 1 µg/ml) treatment protocols have been described (4, 5, 6, 18, 21, 37). Briefly, adherent cells (dish size and number as indicated in figure legends) were stimulated in binding buffer [consisting of 25 mM Tris-HCl (pH 7.4), 120 mM NaCl, 5 mM KCl, 1.2 mM MgCl2, 0.1% (wt/vol) BSA, and 1 mM dextrose] or DMEM (low glucose) with 0.5% (wt/vol) BSA. Stimulations were terminated by washing the cells once with and then harvesting by scraping in ice-cold PBS in the presence of 0.4 mM sodium orthovanadate (PBS-vanadate). Pelleted cells were collected by brief centrifugation. For each cell type, pelleted cells were solubilized for 15 min at 4 C in fusion lysis buffer [1% (vol/vol) Triton X-100, 150 mM NaCl, 10% (vol/vol) glycerol, 50 mM Tris-HCl (pH 8.0), 100 mM NaF, 2 mM EDTA, 1 mM phenylmethylsulfonylfluoride, 1 mM sodium orthovanadate, 10 mM benzamidine, 10 µ g/ml aprotinin], as indicated. After centrifugation at 15,000 x g for 15 min at 4 C, the detergent extracts were electrophoresed under reducing conditions or subjected to immunoprecipitations, as indicated.

For immunoprecipitation of the GHR with the monoclonal anti-GHRcyt-MAb antibodies, 0.6 µg of purified antibody was used per precipitation. Protein-A Sepharose (Pharmacia Biotech, Piscataway, NJ) was used to adsorb immune complexes. For deglycosylation of rbGHR mutants, immunoprecipitates were eluted, heated (at 95 C for 10 min) and treated with endoH (New England Biolabs) or vehicle in accordance with previously published methods (7, 23, 24) and the manufacturer’s suggestions. Sodium dodecyl sulfate sample buffer eluates were resolved by SDS-PAGE and immunoblotted as indicated.

Resolution of proteins by SDS-PAGE, Western transfer of proteins, and blocking of Hybond-ECL (Amersham Pharmacia Biotech, Arlington Heights, IL) with 2% BSA were performed as previously described (4, 5, 6, 18). Immunoblotting with antibodies 4G10 (1:4,000), anti-GHRcyt-AL47 (1:2,000), anti-Myc (1:2,000), anti-pJAK2 (1:1,000), or anti-JAK2AL33 (1:1,000), with horseradish peroxidase-conjugated antimouse or antirabbit secondary antibodies (1:15,000) and detection reagents (SuperSignal West Pico Chemiluminescent Substrate) (all from Pierce Chemical Co., Rockford, IL) and stripping and reprobing of blots were accomplished according to the manufacturer’s suggestions.

Purification and N-Terminal Sequencing of GHR Remnant
High-titered adenovirus stock encoding mGHR1-301-Myc-His, described above, was used to infect thirty 150 x 25 mm dishes of HEK-293 cells. After 24 h, the medium was removed and serum starvation was initiated. After 18 h, the cells were treated with PMA (1 µg/ml, final) for 30 min and then harvested and lysed with fusion lysis buffer, modified to lack EDTA. This detergent cell extract was applied to a TALON Metal Affinity Resin (Co++-TC-Sepharose) column (15 ml). The column was washed twice with 50 ml wash buffer [PBS with 0.5% (vol/vol) Triton X-100 and 1 mM phenylmethylsulfonylfluoride], followed by three washes with 10 mM imidazole in PBS. Proteins bound to the column were eluted with 50 ml PBS containing 150 mM imidazole. This eluate was concentrated by Centriprep-10K (Amicon, Inc., Beverly, MA) size exclusion. The concentrated sample was precipitated with ice-cold acetone. Proteins in this concentrate were resolved by SDS-PAGE (15% acrylamide) and electrically transferred to a polyvinylidene difluoride membrane (Millipore Corp., Bedford, MA). A thin strip of the lane containing the purified proteins was cut off and immunoblotted with anti-Myc to verify the location of the band of interest. The remainder of the membrane was stained with Coomassie Brilliant Blue G250, and the Coomassie-stained band corresponding to the specific anti-Myc-identified remnant band was excised from the membrane for N-terminal sequencing by Edman degradation (38) using an Applied Biosystems 494Ht sequencer (Applied Biosystems, Foster City, CA).

GHBP Assay
GHBP activity was measured in conditioned media by a standard GH binding assay, as previously reported (4, 6, 39). Conditioned medium (0.05 or 0.4 ml, as indicated) from cells treated as indicated was incubated with freshly labeled [125I]hGH (~0.5 ng) for 45 min at 37 C. Bound GH was then immediately separated from free GH by gel chromatography on a Sephadex G-100 column at 4 C. The fraction of GH bound was determined by peak integration.

Densitometric Analysis
Densitometric quantitation of immunoblots was performed using a high-resolution scanner (Epson) and the Image 1.49 program (developed by W. S. Rasband, Research Services Branch, National Institute of Mental Health, Bethesda, MD). For normalization of GHBP shedding in the experiments in Fig. 6CGo, the relative abundance of transfected GHRs among transfections within each experiment was estimated by densitometric scanning of the mature GHR form present in the immunoblot (such as in Fig. 6AGo). The measured GHBP shed into the supernatant of each sample was thus corrected by the abundance of receptor expressed within that transfection to facilitate comparison between WT and mutants. For normalization of GHR proteolysis (as in Fig. 6BGo), the densitometrically determined abundance of remnant in each sample was normalized by the densitometrically determined abundance of the mature GHR form in the same sample. This ratio was compared in each case to the same ratio for the WT receptor within the same experiment, which was considered as 100%.

[125I]hGH Cell Surface Binding Assay
To compare the capacities of the WT and mutant rbGHR to bind GH at the cell surface, [125I]hGH binding assays were performed using HEK-293 cells transiently transfected with each GHR cDNA expression construct, as previously described (17). Transfected HEK-293 cells expressing each receptor form were equally divided into multiple wells of a six-well plate. Replicate samples of serum-starved cells were incubated in 1 ml binding buffer with [125I]hGH [38,000 cpm (~15 pM) per well] either in the presence (to determine nonspecific binding) or absence of 2 µg/ml (~91 nM) unlabeled hGH for 1 h at 25 C. Cells were washed twice with PBS and solubilized in 0.5 ml 1% sodium dodecyl sulfate-0.1 N NaOH and the lysate was subjected to {gamma}-counting. To normalize for transfection efficiency, an equal aliquot of each pool of transfected cells was subjected to anti-GHR immunoblotting, and the specifically bound radiolabeled GH was normalized by the densitometrically determined relative abundance of transfected receptor. Data were expressed (in Fig. 4BGo) as [125I]GH binding relative to GHR abundance as a percentage of the value determined for the WT receptor (considered 100%) within the same experiment.

Statistical Analysis
Statistical analysis was performed by ANOVA or Student’s t test, as appropriate.


    ACKNOWLEDGMENTS
 
The authors appreciate helpful conversations with Drs. J. Kudlow, J. Messina, K. Zinn, Y. Huang, K. Loesch, J. Cowan, and N. Yang and the generous provision of reagents by those named in the text.


    FOOTNOTES
 
This work was supported by Veterans Affairs Merit Review awards (to S.J.F. and to G.B.), a grant from the National Science Foundation (to G.B.), and in part by NIH Grants DK46395 and DK58259 (to S.J.F.).

Parts of this work were presented at the 84th and 85th Annual Meetings of The Endocrine Society in San Francisco, CA, 2002, and Philadelphia, PA, 2003, respectively.

Abbreviations: anti-pTyr, Antiphosphotyrosine; ECD, extracellular domain; endoH, endoglycosidase; GHBP, GH binding protein; GHR, GH receptor; HEK, human embryonic kidney; hGHR, human GHR; IC3, Immunex compound 3; JAK, Janus kinase; mGHR, mouse GHR; PMA, phorbol 12-myristate 13-acetate; rbGHR, rabbit GHR; TACE, TNF{alpha}-converting enzyme; TMD, transmembrane domain; WT, wild-type.

1 We have adhered in this manuscript to the GHR numbering conventions adopted by us and others in the GHR field. Numbering for the rbGHR and hGHR begins with residue 1 being the first predicted residue resulting after signal peptide cleavage; thus these receptors are 620 residues, rather than 638 residues, in length. In contrast, numbering for the mGHR and rGHR begins with residue 1 being the methionine encoded by the initial AUG codon in the GHR mRNA. We have adhered to these disparate numbering systems between rbGHR and mGHR to make comparisons to our previous rbGHR mapping and mutagenesis study (7 ) and other studies easier for the reader. Back

Received for publication April 4, 2003. Accepted for publication June 19, 2003.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Frank SJ, Messina JL 2002 Growth hormone receptor. In: Oppenheim JJ, Feldman M, eds. Cytokine reference on-line. London: Academic Press, Harcourt
  2. Argetsinger LS, Campbell GS, Yang X, Witthuhn BA, Silvennoinen O, Ihle JN, Carter-Su C 1993 Identification of JAK2 as a growth hormone receptor-associated tyrosine kinase. Cell 74:237–244[Medline]
  3. Carter-Su C, Schwartz J, Smit LS 1996 Molecular mechanism of growth hormone action. Annu Rev Physiol 58:187–207[CrossRef][Medline]
  4. Alele J, Jiang J, Goldsmith JF, Yang X, Maheshwari HG, Black RA, Baumann G, Frank SJ 1998 Blockade of growth hormone receptor shedding by a metalloprotease inhibitor. Endocrinology 139:1927–1935[Abstract/Free Full Text]
  5. Zhang Y, Jiang J, Black RA, Baumann G, Frank SJ 2000 TACE is a growth hormone binding protein (GHBP) sheddase: the metalloprotease TACE/ADAM-17 is critical for (PMA-induced) growth hormone receptor proteolysis amd GHBP generation. Endocrinology 141:4342–4348[Abstract/Free Full Text]
  6. Guan R, Zhang Y, Jiang J, Baumann CA, Black RA, Baumann G, Frank SJ 2001 Phorbol ester- and growth factor-induced growth hormone (GH) receptor proteolysis and GH-binding protein shedding: relationship to GH receptor down-regulation. Endocrinology 142:1137–1147[Abstract/Free Full Text]
  7. Wang X, He K, Gerhart M, Huang Y, Jiang J, Paxton RL, Yang S, Lu C, Menon RK, Black RA, Baumann G, Frank SJ 2002 Metalloprotease-mediated GH receptor proteolysis and GHBP shedding: determination of extracellular domain stem region cleavage site. J Biol Chem 277:50510–50519[Abstract/Free Full Text]
  8. Baumann G, Frank SJ 2002 Metalloproteinases and the modulation of growth hormone signalling. J Endocrinol 174:361–368[Abstract/Free Full Text]
  9. Baumbach WR, Horner DL, Logan JS 1989 The growth hormone-binding protein in rat serum is an alternatively spliced form of the rat growth hormone receptor. Genes Dev 3:1199–1205[Abstract]
  10. Smith WC, Kuniyoshi J, Talamantes F 1989 Mouse serum growth hormone (GH) binding protein has GH receptor extracellular and substituted transmembrane domains. Mol Endocrinol 3:984–990[Abstract]
  11. Sadeghi H, Wang BS, Lumanglas AL, Logan JS, Baumbach WR 1990 Identification of the origin of the growth hormone-binding protein in rat serum. Mol Endocrinol 4:1799–1805[Abstract]
  12. Baumann G 2001 Growth hormone binding protein. J Pediatr Endocrinol Metab 14:355–375[Medline]
  13. Leung DW, Spencer SA, Cachianes G, Hammonds RG, Collins C, Henzel WJ, Barnard R, Waters MJ, Wood WI 1987 Growth hormone receptor and serum binding protein: purification, cloning and expression. Nature 330:537–543[CrossRef][Medline]
  14. Lu C, Schwartzbauer G, Sperling MA, Devaskar SU, Thamotharan S, Robbins PD, McTiernan CF, Liu J-L, Jiang J, Frank SJ, Menon RK 2001 Demonstration of direct effects of growth hormone on neonatal cardiomyocytes. J Biol Chem 276:22892–22900[Abstract/Free Full Text]
  15. Dastot F, Sobrier ML, Duquesnoy P, Duriez B, Goossens M, Amselem S 1996 Alternatively spliced forms in the cytoplasmic domain of the human growth hormone (GH) receptor regulate its ability to generate a soluble GH-binding protein. Proc Natl Acad Sci USA 93:10723–10728[Abstract/Free Full Text]
  16. Ross RJ, Esposito N, Shen XY, Von Laue S, Chew SL, Dobson PR, Postel-Vinay MC, Finidori J 1997 A short isoform of the human growth hormone receptor functions as a dominant negative inhibitor of the full-length receptor and generates large amounts of binding protein. Mol Endocrinol 11:265–273[Abstract/Free Full Text]
  17. Frank SJ, Gilliland G, Kraft AS, Arnold CS 1994 Interaction of the growth hormone receptor cytoplasmic domain with the JAK2 tyrosine kinase. Endocrinology 135:2228–2239[Abstract]
  18. Zhang Y, Guan R, Jiang J, Kopchick JJ, Black RA, Baumann G Frank SJ 2001 Growth hormone (GH)-induced dimerization inhibits phorbol ester-stimulated GH receptor proteolysis. J Biol Chem 276:24565–24573[Abstract/Free Full Text]
  19. Allevato G, Billestrup N, Goujon L, Galsgaard ED, Norstedt G, Postel-Vinay MC, Kelly PA, Nielsen JH 1995 Identification of phenylalanine 346 in the rat growth hormone receptor as being critical for ligand-mediated internalization and down-regulation. J Biol Chem 270:17210–17214[Abstract/Free Full Text]
  20. Govers R, tenBreke T, van Kerkhof P, Schwartz AL, Strous GJ 1999 Identification of a novel ubiquitin conjugation motif, required for ligand-induced internalization of the growth hormone receptor. EMBO J 18:28–36[Abstract/Free Full Text]
  21. Zhang Y, Jiang J, Kopchick JJ, Frank SJ 1999 Disulfide linkage of growth hormone receptors reflects GH-induced GHR dimerization: association of JAK2 with the GHR is enhanced by receptor dimerization. J Biol Chem 274:33072–33084[Abstract/Free Full Text]
  22. de Vos AM, Ultsch M, Kossiakoff AA 1992 Human growth hormone and extracellular domain of its receptor: crystal structure of the complex. Science 255:306–312[Medline]
  23. Silva CM, Day RN, Weber MJ, Thorner MO 1993 Human growth hormone (GH) receptor is characterized as the 134-kilodalton tyrosine-phosphorylated protein activated by GH treatment in IM-9 cells. Endocrinology 133:2307–2313[Abstract]
  24. Yi W, Kim S-O, Jiang J, Park SH, Kraft AS, Waxman DJ, Frank SJ 1996 Growth hormone receptor cytoplasmic domain differentially promotes tyrosine phosphorylation of STAT5b and STAT3 by activated JAK2 kinase. Mol Endocrinol 10:1425–1443[Abstract]
  25. Baumann G, Amburn K, Shaw MA 1988 The circulating growth hormone (GH)-binding protein complex: a major constituent of plasma GH in man. Endocrinology 122:976–984[Abstract]
  26. Lim L, Spencer SA, McKay P, Waters MJ 1990 Regulation of growth hormone (GH) bioactivity by a recombinant human GH-binding protein. Endocrinology 127:1287–1291[Abstract]
  27. Mannor DA, Winer LM, Shaw MA, Baumann G 1991 Plasma growth hormone (GH)-binding proteins: effect on GH binding to receptors and GH action. J Clin Endocrinol Metab 73:30–34[Abstract]
  28. Hansen BS, Hjorth S, Welinder BS, Skriver L, De Meyts P 1993 The growth hormone (GH)-binding protein cloned from human IM-9 lymphocytes modulates the down-regulation of GH receptors by 22- and 20-kilodalton human GH in IM-9 lymphocytes and the biological effects of the hormone in Nb2 lymphoma cells. Endocrinology 133:2809–2817[Abstract]
  29. Clark RG, Mortensen DL, Carlsson LM, Spencer SA, McKay P, Mulkerrin M, Moore J, Cunningham BC 1996 Recombinant human growth hormone (GH)-binding protein enhances the growth-promoting activity of human GH in the rat. Endocrinology 137:4308–4315[Abstract]
  30. Sotiropoulos A, Goujon L, Simonin G, Kelly PA, Postel-Vinay MC, Finidori J 1993 Evidence for generation of the growth hormone binding protein through proteolysis of the growth hormone membrane receptor. Endocrinology 132:1863–1865[Abstract]
  31. Dastot F, Duquesnoy P, Sobrier M-L, Goossens M, Amselem S 1998 Evolutionary divergence of the truncated growth hormone receptor isoform in its ability to generate a soluble growth hormone binding protein. Mol Cell Endocrinol 137:79–84[CrossRef][Medline]
  32. Black RA, Durie FH, Otten-Evans C, Miller R, Slack JL, Lynch DH, Castner B, Mohler KM, Gerhart M, Johnson RS, Itoh Y, Okada Y, Nagase H 1996 Relaxed specificity of matrix metalloproteinases (MMPS) and TIMP insensitivity of tumor necrosis factor-{alpha} (TNF-{alpha}) production suggest the major TNF-{alpha} converting enzyme is not an MMP. Biochem Biophys Res Commun 225:400–405[CrossRef][Medline]
  33. Jin G, Huang Z, Black R, Wolfson M, Rauch C, McGregor H, Ellestad G, Cowling R 2002 A continuous fluorimetric assay for tumor necrosis factor-{alpha} converting enzyme. Anal Biochem 302:269–275[CrossRef][Medline]
  34. Mohler KM, Sleath PR, Fitzner JN, Cerretti DP, Alderson M, Kerwar SS, Torrance DS, Otten-Evans C, Greenstreet T, Weerawarna K, Dronheim SR, Petersen M, Gerhart M, Kozlosky CJ, March CJ, Black RA 1994 Protection against a lethal dose of endotoxin by an inhibitor of tumour necrosis factor processing. Nature 370:218–220[CrossRef][Medline]
  35. Hardy S, Kitamura M, Harris-Stansil T, Dai Y, Phipps ML 1997 Construction of adenovirus vectors through Cre-lox recombination. J Virol 71:1842–1849[Abstract]
  36. Jiang J, Liang L, Kim S-O, Zhang Y, Mandler R, Frank, SJ 1998 Growth hormone-dependent tyrosine phosphorylation of a GH receptor-associated high molecular weight protein immunologically related to JAK2. Biochem Biophys Res Commun 253:774–779[CrossRef][Medline]
  37. Goldsmith, JF, Lee, SJ, Jiang J, Frank, SJ 1997 Growth hormone induces detergent-insolubility of GH receptors in IM-9 cells. Am J Physiol 273:E932–E941
  38. Matsudaira P 1987 Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J Biol Chem 262:10035–10038[Abstract/Free Full Text]
  39. Baumann G, Shaw MA, Amburn K 1989 Regulation of plasma growth hormone-binding proteins in health and disease. Metabolism 38:683–689[Medline]