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.
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ABSTRACT |
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INTRODUCTION |
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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-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.
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RESULTS |
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We first tested the expression and proteolytic cleavage of mGHR1-301-Myc-His in adenovirally infected HEK-293 cells (Fig. 1). 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 6590 kDa, consistent with its being a glycoprotein (Fig. 1A
, bracket). Treatment with PMA caused the enhanced appearance of an additional anti-Myc-reactive band of Mr roughly 16 kDa (Fig. 1A
, 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. 1B
). 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).
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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 297406. 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. 3. rbGHR-
237239 and rbGHR-237239AAA are two previously characterized mutants that harbor either deletion of residues S237PF or replacement of these residues with alanines, respectively. rbGHR-
237239 does not undergo proteolysis or GHBP shedding, whereas rbGHR-237239AAA 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. 2
. 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.
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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. 4B). 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. 5). 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-
237239 and rbGHR-237239AAA (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.
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We quantified data derived from multiple experiments, such as those shown in Fig. 6A, 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. 6B
, 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-
237239, 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-237239AAA, 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. 6C
, 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.
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DISCUSSION |
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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-237239, resulted in complete abrogation of receptor proteolysis and GHBP shedding. Interestingly, replacement of these same residues with alanine (rbGHR-237239AAA) 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
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 metalloproteases 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.
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MATERIALS AND METHODS |
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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 manufacturers 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 297406 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-237239 and rbGHR-237239AAA, 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. 3
. 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 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 271620 [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 271620 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 1620 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 manufacturers 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 manufacturers 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. 6C, 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. 6A
). 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. 6B
), 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
-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. 4B
) 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 Students t test, as appropriate.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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-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.
Received for publication April 4, 2003. Accepted for publication June 19, 2003.
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REFERENCES |
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