A ß-Turn Endocytic Code Is Required for Optimal Internalization of the Growth Hormone Receptor but Not for {alpha}-Adaptin Association

Lieve Vleurick, Alain Pezet, Eduard R. Kühn, Eddy Decuypere and Marc Edery

INSERM U344 (L.V., A.P., M.E.) Faculté de Médecine Necker F-75730 Paris, Cedex 15, France
Leuven Poultry Research Group Zoological Institute (L.V., E.R.K.) and Department of Animal Sciences (E.D.) Katholieke Universiteit Leuven B-3000 Leuven, Belgium


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Intracellular trafficking of GH and its receptor, more particularly the chicken GH receptor (cGHR), was examined in COS-7 cells using biochemical and structural studies. Internalization of radioactive GH by the cGHR is reduced as compared with the rat GHR. On the contrary, activation of gene transcription through Janus kinase-2 was similar for both species. Secondary structures of the cytoplasmic domain of chicken and rat GHR were compared, since ß-turns were reported as internalization signals. The substitution of Pro335-Asp336, present in mammalian GH receptors, with Thr307-Gln308 in the cGHR leads to the loss of a ß-turn within a conserved cytoplasmic region. Mutational analysis indicated that the lower rate of internalization of cGHR, as compared with mammalian GHR, was due to this motif. Our data further show that {alpha}-adaptin, a subunit of adaptor protein AP-2, associates with the GHR upon hormone stimulation. The clathrin-coated pit pathway therefore seems to be involved in the endocytosis of cGHR, as AP-2 is known to intervene in the recruitment of receptors to these pits. Interaction with {alpha}-adaptin may occur through a common epitope of the chicken and mammalian GHR, since receptors from both species bind similar amounts of {alpha}-adaptin; alternatively, two different epitopes with similar affinity may be involved. Therefore, not {alpha}-adaptin but an uncharacterized factor, presumably interacting with the identified ß-turn endocytic code, is responsible for the difference in internalization kinetics. Finally, the present study illustrates that functional amino acid motifs of receptors can be derived from comparative studies.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
GH is an important regulator of growth and differentiation in a variety of tissues and species. The receptor mediating GH action (GHR) belongs to the hematopoietic receptor superfamily (1). In addition to the transmembrane GHR, a soluble GH-binding protein (GHBP) has been identified, first in mammals and later in chickens (1, 2). Dimerization of a single GH molecule with two receptors appears to be the first step in hormone-receptor interaction (3). Subsequently, a cytoplasmic tyrosine kinase, Janus kinase 2 (JAK2), associates with each GHR, and JAK2 and GHR molecules are tyrosyl phosphorylated (4). Further downstream the signal transduction pathway, proteins that couple ligand binding to activation of gene transcription, signal transducers and activators of transcription (STAT proteins), are phosphorylated (1).

A characteristic of receptors for polypeptide hormones is their ability for receptor-mediated endocytosis (5). Internalization starts with the recruitment of hormone-receptor complexes into clathrin-coated pits by AP-2, a plasma membrane-specific, heterotetrameric adaptor protein (6). Several cytoplasmic motifs, called endocytic codes or internalization signals, are required for efficient internalization and/or AP-2 association (7, 8). Studies with rat GHR (rGHR) deletion mutants have indicated that the first 90 amino acid (aa) residues of the cytoplasmic domain contain endocytic codes, of which Phe346 has been identified (9). A Leu-pair and a tetrapeptide predicted to adopt a ß-turn were suggested to be internalization signals for the short isoform of the rat PRL receptor (srPRLR) (10). Ligand-mediated endocytosis, however, appears not to be a prerequisite for GH-induced gene transcription mediated by the JAK/STAT signal transduction pathway (9).

Our interest in the internalization of GH and GHR arose from studies in which hepatic GHR capacity was reduced after injecting GH into hypophysectomized chickens (11). Fractionation experiments of adult hen liver indicated that a major portion of GHR was allocated to an intracellular compartment (12). A detailed in vitro study of internalization kinetics, the first in a nonmammalian species, became possible by the cloning of the chicken GHR (cGHR) (13). Moreover, analysis of the GHR amino acid sequence suggested that an endocytic code identified in the srPRLR (10) was present in mammalian GH receptors but not in the cGHR, making the cGHR an interesting model for the study of receptor-mediated endocytosis.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Characterization of Binding to cGHR
COS-7 cells were transiently transfected using the cDNA encoding the full-length cGHR cloned in pSG5, a eukaryotic expression vector. Results from specific binding of [125I]cGH and [125I]hGH to whole cells are presented in Fig. 1Go. By Scatchard analysis, properties of binding were calculated. Since the heterologous human GH (hGH) is a better competitor of [125I]cGH than cGH, [125I]hGH was used for further experiments. Dissociation constant (Kd) for hGH is 0.50 ± 0.06 nM (n = 4), which is within the range reported for binding to the GHR, in a similar transfection study or in hepatic microsomal fractions (14, 15, 16). On the average, 1,000,000 binding sites for hGH are found per transfected cell.



View larger version (18K):
[in this window]
[in a new window]
 
Figure 1. Characterization of Binding to cGHR

COS-7 cells were transiently transfected with 50 ng cGHR cDNA. Intact cells were incubated with [125I]GH and varying concentrations of unlabeled GH. Cells were incubated overnight at 4 C. At the end of incubation, cell-bound radioactivity was recovered by lysis with 1 N NaOH. a, Competition of [125I]cGH from the cGHR by a range of unlabeled cGH (•) or hGH ({circ}) concentrations. b, The data in panel a were used to perform Scatchard analysis.

 
Internalization of GH by cGHR and rGHR
To examine the kinetics of GH uptake, we compared the internalization of [125I]hGH in COS-7 expressing the cGHR or rGHR (Fig. 2Go). At t = 0 min, cell surface binding values for rGHR and cGHR are identical, 17% and 20%, respectively. Internalization by the cGHR is slower as compared with the rGHR. After 15 min at 37 C, label internalized through the cGHR is barely detectable, whereas internalization in the rGHR-transfected cells has already started (P < 0.01). After 1 h at 37 C, internalization by the cGHR is 40% lower than by the rGHR (P < 0.0001). When cGHR-transfected cells are left longer at 37 C, the curve flattens and does not attain rat maximal levels (results not shown). Labeled hGH and cGH are internalized equally by the cGHR (results not shown).



View larger version (17K):
[in this window]
[in a new window]
 
Figure 2. Internalization of Surface-Bound GH by cGHR or rGHR

COS-7 cells expressing cGHR (•) or rGHR ({circ}) were incubated with [125I]hGH at 4 C, washed, and subsequently transferred to 37 C to start internalization. At various time points, cells were washed with acidic buffer to remove surface-bound radioactivity and lysed to determine intracellular radioactivity. Internalization is expressed as the percentage of specific bound counts at time 0 and is corrected for nonspecific internalization. Each point represents the mean (±SEM) of duplicate measurements in four to six independent experiments.

 
Secondary Structure Prediction of GHR Amino Acid Sequences
To further investigate the mechanism behind the reduced internalization rate of cGHR as compared with rGHR, the primary and secondary structures of the first 90 residues from the cytoplasmic domain of both sequences were compared (Fig. 3Go). In the rGHR, seven tetrapeptides are predicted to adopt a ß-turn configuration by the Chou-Fasman algorithm (17). In the rGHR, as well as in the cGHR, the first ß-turn is predicted with the highest probability and ends in two vicinal Leu (Asp311-Pro312-Asp313-Leu314-Leu315). The next three ß-turns are positioned between aa 330 and 344 and are part of one region contiguous with Phe346. However, the middle ß-turn of these three is not present in chicken due to a triple mutation. In this ß-turn, a Pro is in second place in the rGHR, which, of all amino acids, has the strongest ß-turn potential on this position. Consequently, replacing Pro335 by Thr307 reduces ß-turn potential of the cGHR. As the third residue Asp336 is changed to Gln308, a ß-turn becomes even more improbable. The fourth residue does not affect ß-turn potential.



View larger version (26K):
[in this window]
[in a new window]
 
Figure 3. Predicted Potential for ß-Turn Formation of the Cytoplasmic Domain of cGHR and rGHR

Predicted potential for a ß-turn encompassing four residues starting at position i is calculated from the product of bend frequencies (f): p(t)= fi x fi+1 x fi+2 x fi+3. The conformational parameters Pt, P{alpha}, and Pß, express the relative frequency of a specific amino acid to occur in a ß-turn, {alpha}-helix, or ß-sheet, respectively, and are averaged over the four residues ({Pt}, {P{alpha}} and {Pß}). A ß-turn is predicted if p(t) > 0.75 x 10-4, {Pt} > 1, {P{alpha}} < {Pt} > {Pß}, and pi-1(t) < pi(t) > pi+1(t). The signal peptide (16 amino acids) is included when numbering residues. The extracellular domain of the cGHR is 28 amino acids shorter as compared with mammalian GHRs due to the absence of the exon 3 homolog of the hGHR (15 ). a, rGHR. b, cGHR.

 
Characterization of Flag-Tagged cGHR Construct
To enable immunoprecipitation and immunodetection of the cGHR with high efficiency, we added an 8-aa-long Flag epitope to the N terminus of the mature cGHR (Flag-cGHR). A cDNA fragment encoding the cGHR was generated by PCR and subcloned into the expression vector pFlag-CMV-1, immediately after the Flag epitope. For functional studies (activation of gene transcription, JAK/STAT pathway), cytomegalovirus (CMV) promoter-containing plasmids are usually transfected in 293 cells, because they give consistent results in our laboratory (18, 19, 20, 21) as well as other laboratories (22, 23). On the other hand, since COS cells have been previously used to study GHR internalization, we used this cell line for internalization studies. It has been established that simian virus 40 (SV40) promoter-containing plasmids are usually transfected in this cell line because it contains the large T antigen. We did not find a significant difference between the level of GHR expressed using SV40 or CMV promoters in COS cells as determined by Scatchard analysis (Table 1Go), in agreement with previous results (20). Signal transduction and activation of transcription are similar for the wild-type and Flag-tagged GHR as well. GH-dependent induction of luciferase activity is used to assay bioactivity. Furthermore, indirect immunofluorescence studies using Flag-cGHR-transfected cells showed that the epitope-tagged receptor is internalized to cell compartments that participate in the endocytic pathway (data not shown). Indeed, previous studies with N- and C-terminally Flag-tagged PRLR have shown that epitope tagging does not significantly change bioactivity, binding properties, or internalization behavior (24).


View this table:
[in this window]
[in a new window]
 
Table 1. Expression and Functional Activity of the Wild-Type and Flag-Tagged Chicken GHR

 
GH-Dependent Induction of Phosphorylation of JAK-2 and STAT5
The cGHR activates the JAK/STAT pathway, as indicated by the GH-dependent induction of the luciferase reporter gene (Table 1Go). Phosphorylation on tyrosine residues of JAK2 and STAT5 was analyzed for the rat and chicken GHR (Fig. 4Go). JAK2- and STAT5-containing complexes were precipitated from cell lysates using specific anti-JAK2 or anti-STAT5 antibodies. Tyrosine phosphorylation was then evaluated by SDS-PAGE in reducing conditions, followed by probing of the obtained blots with a specific monoclonal antiphosphotyrosine antibody. Tyrosyl phosphorylation of JAK2 (Mr 130,000) is induced by GH, as early as 15 min after GH addition. Later on, GH-dependent tyrosyl phosphorylation of JAK2 decreases. Similarly, activation of STAT5 (Mr 95,0000) by GH is maximal at 15 min. Although internalization of the cGHR is reduced, this does not inhibit the initial steps of signal transduction by the JAK/STAT pathway, i.e. activation of tyrosine kinase JAK2 and of STAT5 during the same time course. On the contrary, tyrosyl phosphorylation of STAT5 appears prolonged for the cGHR, as compared with rabbit GHR (rbGHR).



View larger version (28K):
[in this window]
[in a new window]
 
Figure 4. GH-Induced Tyrosine Phosphorylation of JAK2 and STAT5 by cGHR and rGHR

Cells, transiently expressing GHR, were stimulated or not with 50 nM hGH for 15–60 min; whole-cell lysates were prepared; an anti-JAK2 or anti-STAT5 antibody was used for immunoprecipitation (IP); precipitates were subsequently analyzed by SDS-PAGE; proteins transferred to membrane and blots were probed with antiphosphotyrosine ({alpha}-PY). In parallel experiments, the antibody used for IP was used for blotting, identifying the induced protein band and indicating that similar amounts of JAK2 and STAT5 proteins are present in each lane.

 
Involvement of {alpha}-Adaptin in GHR Internalization
To establish the involvement of coated pits in endocytosis by the cGHR, we used Western blot analysis to study the interaction between cGHR and {alpha}-adaptin, a component of adaptor protein AP-2 (Fig. 5Go). COS-7 cells expressing either Flag-cGHR or a mammalian GHR [Flag-tagged rbGHR (Flag-rbGHR)] were analyzed for their association with {alpha}-adaptin upon hormone stimulation. Immunoblot analysis of Flag-GHR complexes that were purified using immunoprecipitation with an anti-Flag antibody reveals that a specific band of apparent Mr of 100,000 corresponds to {alpha}-adaptin and that it displays a GH-dependent association with the GHR. The amount of {alpha}-adaptin associated with the cGHR is at least as large as that associated with the rbGHR.



View larger version (41K):
[in this window]
[in a new window]
 
Figure 5. Coprecipitation of {alpha}-Adaptin and Flag-GHR

COS-7 cells, transfected with the Flag-tagged GHR vector, were stimulated (+) or not (-) with 45 nM hGH for 20 min; an anti-Flag monoclonal antibody M1 was used for immunoprecipitation of receptor complexes from cell lysates; receptor and associated proteins were subsequently separated by SDS-PAGE, transferred to membrane, and incubated with different antibodies. A mol wt ladder is indicated on the left. a, Coprecipitation of GH receptors and {alpha}-adaptin. To minimally disturb receptor complexes, agarose beads were rinsed only briefly. Membranes were incubated with a monoclonal {alpha}-adaptin antibody. b, Western blot analysis of GHRs with anti-Flag monoclonal antibody M2.

 
Internalization Study of rGHR and cGHR Mutants
The involvement in receptor-mediated endocytosis of the ß-turn identified in previous experiments with the cGHR and rGHR was further tested by mutational analysis. A mutant Flag-cGHR was constructed in which the defective ß-turn was replaced by the respective residues present in the rGHR (Thr307-Gln308 -> Pro307-Asp308). Mutation of the potential endocytic code did not affect binding characteristics of hGH (Kd, 0.4 nM; 21% specific binding; on the average 910,000 binding sites for hGH are found per transfected cell). Notwithstanding the different endocytic behavior of the rat and wild-type chicken GHR, internalization rates of the mutant cGHR and the rGHR were similar, confirming the relevance of the mutated ß-turn for receptor-mediated endocytosis of GH (Fig. 6Go). Activation of gene transcription by the JAK/STAT pathway was identical (luciferase activity induction factor of 9.1 ± 0.8 for wild-type cGHR vs. 9.5 ± 0.5 for Pro307-Asp308 cGHR mutant). The association of the mutant to {alpha}-adaptin was similar to that of wild-type cGHR (data not shown). Furthermore, the pivotal role of Pro335 for optimal internalization of the rGHR was corroborated by alanine scanning of the Lys334-Pro335-Asp336-Phe337 tetrapeptide in the rGHR. The Pro335-> Ala335 rGHR mutant internalized less (t = 60 min, P < 0.01) than Lys334-> Ala334, Asp336-> Ala336, and Phe337-> Ala337 mutants that are very similar in internalization compared with the wild-type rGHR (Fig. 7Go).



View larger version (16K):
[in this window]
[in a new window]
 
Figure 6. Internalization of Surface-Bound GH by Wild-Type or P307D308 Mutated cGHR

See Fig. 3Go for protocol details. Each point represents the mean (±SEM) of duplicate measurements in three independent experiments.

 


View larger version (22K):
[in this window]
[in a new window]
 
Figure 7. Internalization of Surface-Bound GH by rGHR Mutants Generated by Alanine Scanning of the Putative ß-Turn Internalization Motif

See Fig. 3Go for protocol details. Each point represents the mean (±SEM) of duplicate measurements in four independent experiments.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In this report, kinetics and mechanisms of GH internalization by receptor-mediated endocytosis are described. In the absence of a conserved ß-turn in the juxtamembranous intracellular domain, internalization is strongly reduced. Furthermore, a more detailed understanding of endocytosis was obtained by studying the interaction of a GHR with {alpha}-adaptin, a subunit of the AP-2 complex that initiates formation of clathrin-coated pits (6). The identified endocytic code was not involved in ligand binding, {alpha}-adaptin association, or activation of JAK/STAT.

Although internalization of the cGHR, as compared with mammalian GH receptors, was reduced, gene transcription through the JAK/STAT signal transduction pathway was activated by the cGHR. Transcriptional activity mediated by STAT5 was evaluated with a reporter gene. The gene construct contains lactogenic hormone response element (LHRE) coupled to the thymidine kinase minimal promoter and the luciferase gene. LHRE is the element of the ß-casein promoter that was used for affinity purification of STAT5 (25). Moreover, the level and timing of tyrosine phosphorylation of JAK2 of the cGHR and rGHR did not differ. Tyrosyl phosphorylation of STAT5 seemed even prolonged. If the cGHR has a longer half-life on the plasma membrane, then STAT5 has more time to bind to phosphorylated tyrosines within the GHR and will be more phosphorylated by JAK2. Distinction of internalization capacity and signal transduction was previously evaluated at the molecular level; different cytoplasmic regions of the GHR sequence are responsible for these processes. Proximal to the membrane, the GHR contains a Pro-rich region, called Box 1 (aa 297–311 in rGHR), that is conserved in the cytokine/GH/PRL receptor family and is required for signal transduction by the JAK/STAT pathway (16). On the other hand, the cytoplasmic region of the rGHR ranging from amino acid residue 318–380 is involved in internalization. More specifically, Phe346 was identified as being critical for GH-dependent internalization of the rGHR, but not for activation of gene transcription by JAK/STAT (9).

Sequences of internalization signals were first elucidated for the low-density lipoprotein receptor, the transferrin receptor, and the cation-independent mannose-6-phosphate receptor. Although endocytic codes of these receptors differ in specific sequence, they all share a common three-dimensional conformation and chemistry and form tight (or ß-) turns. For the transferrin receptor, it was reported that a Tyr-containing ß-turn internalization signal could be replaced by Leu-Leu, which suggests that these two signals are functionally equivalent (7). Recently it was reported, however, that the di-Leu motif is solely involved in the internalization of truncated GHR or PRLR (srPRLR), but not of the full-length GHR (26).

Secondary structure analysis of the juxtamembranous cytoplasmic region of the GHR with high interspecies homology revealed the presence of several peptide motifs, predicted to adopt a ß-turn configuration, the first tight turn having the highest probability found in our analyzed region. In the region important for efficient internalization by the rGHR (aa 318–380), three contiguous ß-turn tetrapeptides occur within a highly conserved stretch of 15 aa. The middle ß-turn of this trio, however, is not present in chicken, as Pro307 and Asp308 are replaced in cGHR by residues with less ß-turn potential. The fourth residue, Phe309, is not conserved either, but this mutation does not affect ß-turn probability. Moreover, by Ala mutation, Phe309 was previously shown not to be required for internalization (9). Finally, the rGHR Phe346 identified previously as an internalization motif (9) is conserved in the cGHR. The reduced internalization capacity of the cGHR may therefore be due to the different three-dimensional structure of the receptor molecule, which hinders endocytosis. The current vision is that these ß-turn internalization motifs are exposed on the surface of the cytoplasmic tail, enabling interaction with adaptor molecules (7). Deletion of a similar motif (Leu-Pro-Gly-Gly) in the srPRLR caused a 50% reduction in internalization (10), which corresponds to the diminished internalization of cGHR, as compared with rGHR. The remaining internalization capacity of the cGHR could then be due to endocytic codes common to both species.

Little is known about the underlying mechanisms controlling GHR internalization and trafficking. In general, receptor-mediated endocytosis involves the concentration of receptors in clathrin-coated pits with the help of a plasma membrane-associated adaptor protein AP-2. This protein complex consists of four subunits: {alpha}2- and ß-adaptin of 100 kDa, a µ2-chain of 50 kDa, and a small {varsigma}2-chain of 16 kDa (6). Upon ligand stimulation, {alpha}-adaptin associated with the srPRLR (10). Our results demonstrate, for the first time, the GH-inducible binding of a GHR to {alpha}-adaptin.

Furthermore, we compared the interaction of {alpha}-adaptin with chicken or mammalian GHRs, in view of the species-dependent internalization behavior. As both receptors bind similar amounts of {alpha}-adaptin, our experiments indicate that cytoplasmic motifs required for internalization and for {alpha}-adaptin binding are not necessarily the same. Further experiments are necessary to determine which region(s) of the GHR is (are) responsible for {alpha}-adaptin association. In vitro studies showed that Tyr-containing sorting signals interact with the µ2-subunit of AP-2 (27). The existence of distinct motifs for internalization and AP-2 association, respectively, was reported for the receptor of epidermal growth factor. Kinetics of internalization of a mutant receptor for epidermal growth factor lacking the Tyr-containing AP-2 binding site were indistinguishable from those of its wild-type counterpart and were independent of AP-2 (28).

Although there is no doubt that AP-2 is involved in clathrin-dependent endocytosis, the previous and present studies suggest that {alpha}-adaptin may be necessary, but not sufficient, for a maximal response. The ß-turn lost in cGHR may interact with ß-arrestin that induced a concentration of the ß2-adrenergic receptor in clathrin-coated pits (29). Furthermore, mammalian and chicken GHR may differ in ubiquitin association. Recently it was shown that GHR ubiquitination is not only a prerequisite for GHR degradation, but also for ligand-dependent endocytosis (30). Moreover, the ubiquitin conjugation and ligand-induced internalization are coupled events, since they are both disrupted by the F345A mutation (31). Finally, GHBP may interfere with GHR internalization. The culture medium of cells transfected with the cGHR contains large amounts of GHBP, since cGHBP is generated by proteolysis of the full-length GHR (32), contrary to rGHBP that is not detectable in medium of rGHR-transfected cells (33). The formation of GH.GHR.GHBP heterotrimers may hinder internalization of the cGHR.

In conclusion, our study shows that 1) the cGHR is internalized, a process presumably mediated by the clathrin-coated pits endocytic pathway, 2) reduced internalization of the cGHR as compared with mammalian GH receptors is related to the loss of a conserved cytoplasmic internalization motif predicted to adopt a ß-turn, and 3) the level of internalization is not correlated to the level of {alpha}-adaptin association or activation of gene transcription.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Hormones and Expression Vectors
Recombinant hGH was kindly provided by Serano Ares Laboratories (Geneva, Switzerland). Recombinant cGH was a generous gift of Ciba-Geigy (Basel, Switzerland) and natural cGH was a gift of Dr. Luc Berghman (K.U. Leuven, Belgium) (34). [125I]GH was prepared as described previously [hGH (35), recombinant cGH (11)] to a specific activity of 80–140 µCi/µg.

Dr. J. Burnside (University of Delaware, Newark, DE) kindly provided the cDNA encoding the cGHR cloned in the pSG5 expression vector that is under SV40 transcriptional control (36). The rGHR expression plasmid pLM108, a pUC8 construct containing the human metallothionein IIa promoter and SV40 enhancer, was donated by Dr. G. Norstedt (Center for Biotechnology, Karolinska Institute, NOVUM, Huddinge, Sweden) (37).

Construction of Flag-Tagged cGHR
To enhance immunoprecipitation and immunodetection, the cGHR was epitope-tagged at the N terminus with an octapeptide, named Flag (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys). Monoclonal antibodies directed against the Flag-epitope are commercially available (Eastman Kodak Co., Rochester, NY). The cDNA encoding the mature wild-type cGHR was inserted in the pFlag-CMV-1 expression vector (Eastman Kodak Co.), after the preprotrypsin signal peptide sequence that ensures correct membrane-bound expression. By oligonucleotide-directed mutagenesis using PCR, a cGHR insert was produced, excluding the original signal peptide (the first 16 aa) and introducing at the 5'- and 3'-end unique restriction enzyme sites recognized by NotI and XbaI. An N-terminally Flag-tagged cGHR construct (pFlag-cGHR) was obtained after enzyme digestion, ligation, and ampicillin selection in Escheria coli DH5 cells. The presence of the Flag epitope in the engineered cGHR was confirmed by dideoxynucleotide sequence analysis (38). The rbGHR inserted in pFlag-CMV-1 vector (pFlag-rbGHR) was obtained from S. Moutoussamy (INSERM U344) (22).

Construction of Flag-Tagged cGHR (T307P,Q308D) and rGHR Mutants (K334A; P335A; D336A; F337A)
Substitutions of Thr307 and Gln308 by Pro and Asp, respectively, were obtained using the Flag-CMV-1-cGHR plasmid. A single-stranded DNA was generated by using the origin of replication of the M13 phage present in the vector, in the Escheria coli CJ236 strain in the presence of M13K07 helper phage. This single-stranded DNA was then used as a template for oligonucleotide-directed mutagenesis with the primer 5'-CATTGTATAGGTCTGGCTTGTAGTTG-3'. A similar strategy was used to construct K334A, P335A, D336A, and F337A mutants. The modified regions were verified by sequencing (38).

Cell Culture and Transfection
COS-7 cells were grown as monolayers in DMEM containing 10% FCS, 2 mM L-glutamine, 50 IU/ml penicillin, and 50 µg/ml streptomycin. Routinely, cells were cultured at 37 C, in a humid 5% CO2 incubator. At 70–80% confluence, cells were transfected by the diethylaminoethyl-dextran/chloroquine method. Four hours after addition of DNA precipitates, cells were subjected to Me2SO shock, washed, and cultured in fresh complete medium for 48 h (10).

Analysis of GHR Binding
Hormone binding to transfected COS-7 cells was performed in six-well plates. Before binding, cells were washed with DMEM and kept in serum-free medium for at least 4 h. Subsequently, plates were put on ice and washed with ice-cold HEPES binding buffer (HBB: 25 mM HEPES, 124 mM NaCl, 4 mM KCl, 1 mM CaCl2, 1.5 mM MgCl2, and 2 mM KH2PO4, pH 7.4). In a final volume of 1 ml HBB containing 1% BSA (Fraction V, Sigma Chemical Co., St. Louis, MO) (HBB-BSA) whole cells were incubated with 80,000–100,000 cpm [125I]GH. At the end of incubation, unbound label was removed by two HBB washes; 1 ml 1 N NaOH was added, and radioactivity in lysates was measured using a {gamma}-counter. Specific binding was determined by subtracting the amount of [125I]GH bound in the presence of excess unlabeled GH (91 nM). For Scatchard analysis, increasing amounts of unlabeled GH were added to compete with [125I]GH for binding in saturating conditions (overnight at 4 C). With LIGAND software (Elsevier-BioSOFT, Cambridge, UK), Kd and binding capacity were calculated (39). Data are represented as means ± SEM.

Internalization Studies
Internalization was analyzed as described by Allevato et al. (9). Briefly, cells transfected with 0.1–1 µg cDNA were treated as described for binding analysis. Cells were incubated with 70,000–100,000 cpm [125I]GH at 4 C to bind cell surface receptors (hGH for 2.5 h, cGH for 6 h); the low temperature prevented label internalization. Unbound ligand was removed by washing twice with ice-cold HBB; culture plates containing 1 ml HBB-BSA were then transferred to 37 C for various times; surface-bound [125I]GH was removed by a 3-min exposure to an acid wash buffer (150 mM NaCl and 50 mM glycine, pH 2.5), and cells were lysed to recover acid-resistant binding. For each time point, internalization was expressed as the percentage of specific intracellular radioactivity toward specific binding at t = 0 min. Data were statistically analyzed with SAS software (SAS Institute, Inc., Cary, NC) using the general linear model procedure and are represented as means ± SEM.

Secondary Structure Prediction
The occurrence of ß-turns was predicted using the Chou-Fasman algorithm (17). ß-Turns are chain-reversal regions consisting of tetrapeptides. Based on x-ray crystallography data of 29 proteins, the average overall frequencies to be part of an {alpha}-helix (<f{alpha}> = 0.38), a ß-sheet (<fß> = 0.20), or a ß-turn (<ft> = 0.32) were determined. Conformational parameters Pt, P{alpha}, and Pß were obtained for all 20 amino acids, by expressing their frequency (f{alpha}, fß, and ft) relative to the respective average frequency. Strong ß-turn formers are Asn, Gly, and Pro, all with Pt > 1.50. In addition, the bend frequency (f) of each amino acid was calculated for the four positions of the ß-turn. The probability of a bend starting at residue i is then p(t) = fi x fi+1 x fi+2 x fi+3. To predict whether a tetrapeptide is a ß-turn or rather part of an {alpha}-helix coil or ß-sheet, Pt, P{alpha}, and Pß are averaged over the four residues. A ß-turn is predicted if p(t) > 0.75 x 10-4, {Pt} > 1 and {P{alpha}} < {Pt} > {Pß}.

Luciferase Bioassay
To test biological activity of Flag-tagged and wild-type receptors, a 293-cell bioassay was used (40). A 6-well plate was transfected by the calcium phosphate technique using, per well, 33 ng of receptor plasmid, 17 ng of a ß-galactosidase expression vector (pCH110, Pharmacia Biotech, Uppsala, Sweden), and 250 ng of LHRE-tk-Luc. The latter is a construct that contains a GH-responsive promoter [thymidine kinase (tk) minimal promoter and 6 repeats of the LHRE of the ß-casein promoter] fused to the firefly luciferase reporter gene. One day after transfection, cells were incubated overnight with 0 or 23 nM hGH serum-free medium. Cell extracts were prepared and enzyme activities were determined. Activity of ß-galactosidase was used to normalize luciferase expression levels for differences in transfection efficiency.

Immunoprecipition and Western Blot Analysis
COS-7 cells were grown on 100-mm dishes, transfected with Flag-tagged receptor plasmid (1 µg/dish), cultured overnight in serum-free medium, and stimulated (or not) with 45 nM hGH (20 min at 37 C). Cellular proteins were extracted in 1 ml of lysis buffer (50 mM Tris, 2 mM CaCl2, 100 mM NaCl, 8% glycerol, 0.8% Triton X-100, pH 7.6) containing phosphatase and protease inhibitors (1 mM o-Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 5 µg/ml aprotinin, 2 µg/ml leupeptin, 1 µg/ml pepstatin). Lysates were incubated overnight at 4 C with agarose beads bearing Flag monoclonal antibodies (M1) for precipitation of receptor complexes. Immunoprecipitated complexes were washed with fresh cold lysis buffer, boiled in SDS sample buffer, and subjected to 7.5% SDS-PAGE. Proteins were then transferred onto a polyvinylidene difluoride membrane (PVDF, Polyscreen, Dupont NEN, Boston, MA), and blots were incubated for 2 h at room temperature with monoclonal antibodies directed against either Flag (M2, 0.5 µg IgG/ml) or {alpha}-adaptin (AC1-M11, 1:100, Affinity BioReagents, Inc. Neshanic Station, NJ). Finally, membranes were incubated for 1 h with alkaline phosphatase-linked goat antimouse secondary antibody (1:10,000), and proteins were revealed using the Vistra ECF Western blotting system (Amersham Pharmacia Biotech, Little Chalfont, UK).

To evaluate JAK2 activation by GH, 293 cells were cotransfected with GHR (4 µg) and human JAK2 (2 µg) cDNA. For the determination of GH-dependent induction of STAT5 tyrosyl-phosphorylation, cells were transfected with GHR (4 µg/ml, JAK2 (0.1 µg/ml), and STAT5 (2 µg/ml) cDNA. JAK2 and STAT5 expression vectors were kindly provided by Drs. J. Ihle and B. Groner, respectively. Cell lysates were incubated with the designated antibody [anti-JAK2 (1 µg/ml; Upstate Biotechnology, Inc., Lake Placid, NY) or anti-STAT5 (1 µg/ml; Santa Cruz Biotechnology, Inc., Santa Cruz, CA)] and protein A-Sepharose (50%, vol/vol). Finally, immunoprecipitated complexes were analyzed by Western blot, using a monoclonal antiphosphotyrosine (4G10; Upstate Biotechnology, Inc.; 1:4,000), a polyclonal anti-JAK2 antibody (1:5,000), or a monoclonal anti-STAT5 (1:1,000) antibody. Finally, the membranes were incubated with an antirabbit or antimouse IgG-conjugated horseradish peroxidase (1:8,000) and revealed by the enhanced chemiluminesence (ECL) detection system (Amersham Pharmacia Biotech).


    ACKNOWLEDGMENTS
 
The authors are grateful to W. Van Ham for iodinating cGH, Dr. H. Buteau for plentiful advice, and S. Kotanen for help in manuscript preparation.


    FOOTNOTES
 
Address requests for reprints to: Lieve Vleurick, 13 Avenue Maréchal Juin, B-3000 Gembloux, Belgium.

This work was supported by the Fund for Scientific Research-Flanders (Belgium) (F.W.O. G.0235.97).

Received for publication October 19, 1998. Revision received July 3, 1999. Accepted for publication July 26, 1999.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Finidori J, Kelly PA 1995 Cytokine receptor signalling through two novel families of transducer molecules: Janus kinases, and signal transducers and activators of transcription. J Endocrinol 147:11–23[Medline]
  2. Vasilatos-Younken R, Andersen BJ, Rosebrough RW, McMurtry JP, Bacon WL 1991 Identification of circulating growth hormone-binding proteins in domestic poultry: an initial characterization. J Endocrinol 130:115–122[Abstract]
  3. Cunningham BC, Ultsch M, de Vos AM, Mulkerrin MG, Clauser KR, Wells JA 1991 Dimerization of the extracellular domain of the human growth hormone receptor by a single hormone molecule. Science 254:821–825[Medline]
  4. 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]
  5. Posner BI, Bergeron JJM, Josefsberg Z, Khan MN, Khan RJ, Patel BA, Sikstrom RA, Verma AK 1981 Polypeptide hormones: intracellular receptors and internalization. Recent Prog Horm Res 37:539–582[Medline]
  6. Robinson MS 1994 The role of clathrin, adaptors and dynamin in endocytosis. Curr Opin Cell Biol 6:538–544[Medline]
  7. Trowbridge IS, Collawn JF, Hopkins CR 1993 Signal-dependent membrane protein trafficking in the endocytic pathway. Annu Rev Cell Biol 9:129–161[CrossRef]
  8. Kirchhausen T, Bonifacino JS, Riezman H 1997 Linking cargo to vesicle formation: receptor tail interactions with coat proteins. Curr Opin Cell Biol 9:488–495[CrossRef][Medline]
  9. Allevato G, Billestrup N, Goujon L, Galsgaard ED, Norstedt G, Postel Vinay M-C, 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]
  10. Vincent V, Goffin V, Rozakis-Adcock M, Mornon J-P, Kelly PA 1997 Identification of cytoplasmatic motifs required for short prolactin receptor internalization. J Biol Chem 272:7062–7068[Abstract/Free Full Text]
  11. Vanderpooten A, Darras VM, Huybrechts LM, Rudas P, Decuypere E, Kühn ER 1991 Effect of hypophysectomy and acute administration of growth hormone (GH) on GH-receptor binding in chick liver membranes. J Endocrinol 129:275–281[Abstract]
  12. Vleurick L, Van Veldhoven P, Decuypere E, Kühn ER 1998 Intracellular growth hormone receptors in chicken liver. In: Vaudry H, Tonon M-C, Roubos EW, De Loof A (eds) Trends in Comparative Endocrinology and Neurobiology. Ann NY Acad Sci 839:538–540[Free Full Text]
  13. Burnside J, Liou SS, Cogburn LA 1991 Molecular cloning of the chicken growth hormone receptor complementary deoxyribonucleic acid: mutation of the gene in sex-linked dwarf chickens. Endocrinology 128:3183–3192[Abstract]
  14. Kühn ER, Huybrechts LM, Vanderpooten A, Berghman L 1989 A decreased capacity of hepatic growth hormone (GH) receptors and failure of thyrotrophin-releasing hormone to stimulate the peripheral conversion of thyroxine into triiodothyronine in sex-linked dwarf broiler hens. Reprod Nutr Dev 29:461–467[Medline]
  15. Burnside J, Cogburn LA 1993 Molecular biology of the chicken growth hormone receptor. In: Sharp PJ (ed) Avian Endocrinology. Journal of Endocrinology Ltd, Bristol, UK, pp 161–176
  16. Goujon L, Allevato G, Simonin G, Paquereau L, Le Cam A, Clark J, Nielsen JH, Djiane J, Postel-Vinay M-C, Edery M, Kelly PA 1994 Cytoplasmatic sequences of the growth hormone receptor necessary for signal transduction. Proc Natl Acad Sci USA 91:957–961[Abstract]
  17. Chou PY, Fasman GD 1978 Emperical predictions of protein conformation. Annu Rev Biochem 47:251–276[CrossRef][Medline]
  18. Martini J-F, Pezet A, Guezennec CY, Edery M, Postel-Vinay M-C, Kelly PA 1997 Monkey growth hormone (GH) receptor gene expression: evidence for two mechanisms for the generation of the GH binding protein. J Biol Chem 272:1851–1858
  19. Pezet A, Buteau H, Kelly PA, Edery M 1997 The last proline of Box 1 is essential for association with JAK2 and functional activation of the prolactin receptor. Mol Cell Endocrinol 129:199–208[CrossRef][Medline]
  20. Buteau H, Pezet A, Ferrag F, Perrot-Applanat M, Kelly PA, Edery M 1998 N-Glycosylation of the prolactin receptor is not required for activation of gene transcription but is crucial for its cell surface targeting. Mol Endocrinol 12:544–555[Abstract/Free Full Text]
  21. Moutoussamy S, Renaudie F, Lago F, Kelly PA, Finidori J 1998 Grb10 identified as a potential regulator of growth hormone (GH) signaling by cloning of GH receptor target protein. J Biol Chem 273:15906–15912[Abstract/Free Full Text]
  22. Endo TA, Masuhara M, Yokouchi M, Suzuki R, Sakamoto H, Mitsui K, Matsumoto A, Tanimura S, Ohtsubo M, Misawa H, Miyazaki T, Leonor N, Taniguchi T, Fujita T, Kanakura Y, Komiya S, Yoshimura A 1997 A new protein containing an SH2 domain that inhibits JAK kinases. Nature 387:921–924[CrossRef][Medline]
  23. Ali S 1998 Prolactin receptor regulates STAT5 tyrosine phosphorylation and nuclear translocation by two separate pathways. J Biol Chem 273:7709–7716[Abstract/Free Full Text]
  24. Perrot-Applanat M, Gualillo O, Buteau H, Edery M, Kelly PA 1997 Internalization of prolactin receptor and prolactin in transfected cells does not involve nuclear translocation. J Cell Sci 110:1123–1132[Abstract/Free Full Text]
  25. Wakao H, Gouilleux F, Groner B 1994 Mammary gland factor (MGF) is a novel member of the cytokine regulated transcription factor gene family and confers the prolactin response. EMBO J 13:2182–2191[Abstract]
  26. Govers R, van Kerkhof P, Schwartz AL, Strous GJ 1998 Di-leucine-mediated internalization of ligand by a truncated growth hormone receptor is independent of the ubiquitin conjugated system. J Biol Chem 273:16420–16433
  27. Ohno H, Stewart J, Fournier MC, Bosshart H, Rhee I, Miyatake S, Saito T, Gallusser A, Kirchhausen T, Bonifacino JS 1995 Interaction of tyrosine-based sorting signals with clathrin-associated proteins. Science 269:1872–1875[Medline]
  28. Sorkin A, Mazzotti M, Sorkina T, Scotto L, Beguinot L 1996 Epidermal growth factor receptor interaction with clathrin adaptors is mediated by the Tyr974-containing internalization motif. J Biol Chem 271:13377–13384[Abstract/Free Full Text]
  29. Goodman Jr OB, Krupnick JG, Santini F, Gurevich VV, Penn RB, Gagnon AW, Keen JH, Benovic JL 1996 ß-arrestin acts as a clathrin adaptor in endocytosis of the ß2-adrenergic receptor. Nature 383:447–450[CrossRef][Medline]
  30. Strous GJ, van Kerkhof P, Govers R, Ciechanover A, Schwartz AL 1996 The ubiquitin conjugation system is required for ligand-induced endocytosis and degradation of the growth hormone receptor. EMBO J 15:3806–3812[Abstract]
  31. Govers R, van Kerkhof P, Schwartz AL, Strous GJ 1997 Linkage of the ubiquitin-conjugating system and the endocytic-pathway in ligand-induced internalization of the growth hormone receptor. EMBO J 16:4851–4858[Abstract/Free Full Text]
  32. Vleurick L, Kühn ER, Decuypere E, Burnside J, Pezet A, Edery M 1999 Generation of chicken growth hormone-binding proteins by proteolysis. Gen Comp Endocrinol 113:283–289[CrossRef][Medline]
  33. Sotiropoulos A, Goujon L, Simonin G, Kelly PA, Postel-Vinay M-C, Finidori J 1993 Evidence for generation of the growth hormone-binding protein through proteolysis of growth hormone membrane receptor. Endocrinology 132:1863–1865[Abstract]
  34. Berghman LR, Van Beeumen J, Decuypere E, Kühn ER, Vandesande F 1988 One-step purification of chicken growth hormone from a crude pituitary extract by use of a monoclonal immunoadsorbent. J Endocrinol 118:381–387[Abstract]
  35. Kelly PA, Leblanc G, Dijane J 1979 Estimation of total prolactin-binding sites after in vitro desaturation. Endocrinology 104:1631–1638[Medline]
  36. Huang N, Cogburn LA, Agarwal SK, Marks HL, Burnside J 1993 Overexpression of a truncated growth hormone receptor in the sex-linked dwarf chicken: evidence for a splice mutation. Mol Endocrinol 7:1391–1398[Abstract]
  37. Billestrup N, Moldrup A, Serup P, Mathews LS, Norstedt G, Moldrup A, Nielsen JH 1990 Introduction of exogenous growth hormone receptors augments growth hormone-responsive insulin biosynthesis in rat insulinoma cells. Proc Natl Acad Sci USA 87:7210–7214[Abstract]
  38. Sanger F, Nicklen S, Coulsen AR 1977 DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467[Abstract]
  39. Munson PJ, Rodbard D 1980 LIGAND: a versatile computerized approach for characterization of ligand-binding systems. Anal Biochem 107:220–239[Medline]
  40. Sotiropoulos A, Moutoussamy S, Renaudie F, Clauss M, Kayser C, Gouilleux F, Kelly PA, Finidori J 1996 Differential activation of Stat3 and Stat5 by distinct regions of the growth hormone receptor. Mol Endocrinol 10:998–1009[Abstract]