Metalloprotease-mediated GH receptor proteolysis and GHBP shedding. Determination of extracellular domain stem region cleavage site

Growth hormone-binding protein (GHBP) is complexed to a substantial fraction of circulating GH. In humans, rabbits, and other species, GHBP derives from proteolytic shedding of the GH receptor (GHR) extracellular domain. In cell culture studies, stimuli such as phorbol ester, platelet-derived growth factor, or serum induce GHR proteolysis, which concomitantly yields shed GHBP in cell supernatants and a cell-associated cytoplasmic domain-containing GHR remnant. This process is sensitive to metalloprotease inhibition, and genetic reconstitution studies identify tumor necrosis factor-alpha converting enzyme (TACE/ADAM-17), a transmembrane metalloprotease, as a GHR sheddase. Stimuli that induce GHR proteolysis render cells less responsive to GH, but the mechanism(s) of this desensitization is not yet understood. In this study, we mapped the rabbit (rb) GHR cleavage site. We adenovirally expressed a C-terminal epitope-tagged rbGHR lacking most of its cytoplasmic domain, purified the remnant protein induced by the phorbol ester, PMA, and derived the cleavage site by N-terminal sequencing of the purified remnant. The N-terminal sequence, (239)FTCEEDFR(246), matched perfectly the rbGHR and suggests that cleavage occurs eight residues from the membrane in the proximal extracellular domain stem region. Deletion and alanine substitution mutagenesis indicated that, similar to other TACE substrates, the spacing of residues in this region, more than their identity, influences GHR cleavage susceptibility. Further, we determined that PMA pretreatment desensitized a cleavage-sensitive GHR mutant, but not a cleavage-insensitive mutant, to GH-induced JAK2 activation. These results suggest that inducible GHR proteolysis can regulate GH signaling.     

Growth hormone (GH)-induced dimerization inhibits phorbol ester-stimulated GH receptor proteolysis

Growth hormone (GH) initiates its cellular action by properly dimerizing GH receptor (GHR). A substantial fraction of circulating GH is complexed with a high-affinity GH-binding protein (GHBP) that in many species can be generated by GHR proteolysis and shedding of the receptor's ligand-binding extracellular domain. We previously showed that this proteolysis 1) can be acutely promoted by the phorbol ester phorbol 12-myristate 13-acetate (PMA), 2) requires a metalloprotease activity, 3) generates both shed GHBP and a membrane-associated GHR transmembrane/cytoplasmic domain remnant, and 4) results in down-regulation of GHR abundance and GH signaling. Using cell culture model systems, we now explore the effects of GH treatment on inducible GHR proteolysis and GHBP shedding. In human IM-9 lymphocytes, which endogenously express GHRs, and in Chinese hamster ovary cells heterologously expressing wild-type or cytoplasmic domain internal deletion mutant rabbit GHRs, brief exposure to GH inhibited PMA-induced GHR proteolysis (receptor loss and remnant accumulation) by 60-93%. PMA-induced shedding of GHBP from Chinese hamster ovary transfectants was also inhibited by 70% in the presence of GH. The capacity of GH to inhibit inducible GHR cleavage did not rely on JAK2-dependent GH signaling, as evidenced by its continued protection in JAK2-deficient gamma2A rabbit GHR cells. The GH concentration dependence for inhibition of PMA-induced GHR proteolysis paralleled that for its promotion of receptor dimerization (as monitored by formation of GHR disulfide linkage). Unlike GH, the GH antagonist, G120K, which binds to but fails to properly dimerize GHRs, alone did not protect against PMA-induced GHR proteolysis; G120K did, however, antagonize the protective effect of GH. Our data suggest that GH inhibits PMA-induced GHR proteolysis and GHBP shedding by inducing GHR dimerization and that this effect does not appear to be related to GH site 1 binding, GHR internalization, or GHR signaling. The implications of these findings with regard to GH signaling and GHR down-regulation are discussed.     

Reduced proteolysis of rabbit growth hormone (GH) receptor substituted with mouse GH receptor cleavage site

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.     

Role of the growth hormone (GH) receptor transmembrane domain in receptor predimerization and GH-induced activation

The GH receptor (GHR) mediates GH effects by activating the GHR-associated cytoplasmic tyrosine kinase, Janus kinase 2. Recent studies indicate that GHRs exist as dimers independently of GH binding. Some authors suggest that receptor predimerization is mediated by the transmembrane domain (TMD) and that GH binding initiates signaling by triggering changes in the orientation of the two GHRs within the dimer. In this study, we investigate the role of GHR TMD in GH-independent receptor dimerization and ligand-induced activation. We prepared a GHR mutant, GHR(LDLR), in which the TMD is replaced with the TMD of the human low-density lipoprotein receptor (LDLR). The resultant chimera has a TMD two residues shorter than the native GHR TMD; thus, in addition to possessing a different TMD, the altered GHR(LDLR) TMD helical register may change positions of the GHR extracellular domain (ECD) and intracellular domain relative to the TMD when compared with the wild-type (WT) receptor. When each was coexpressed with an intracellular domain-truncated GHR mutant, GHR(1-274-Myc), both WT GHR and GHR(LDLR) were specifically coprecipitated with GHR(1-274-Myc), indicating that the GHR TMD was not required for GHR heterodimerization with GHR(1-274-Myc). We further examined the contribution of the so-called "dimerization interface," a GHR ECD region that is critical for GH-induced signaling, to receptor predimerization. Coimmunoprecipitation experiments with either WT GHR, a dimerization interface mutant (GHR-H150D), or a control mutant (GHR-T147D) with GHR(1-274-Myc) showed dramatically reduced coprecipitation of GHR-H150D with GHR(1-274-Myc) when compared with WT GHR or GHR-T147K. This result suggests that, in contrast to some recent models, the dimerization interface contributes to GHR predimerization. We also compared WT GHR with GHR(LDLR) and GHR(LDLRDelta4) (a chimera in which the LDLR TMD has an internal deletion of four residues) with regard to response to GH stimulation. Although the chimeras had similar GH dose responses and time courses for signaling as WT GHR, they were markedly less sensitive to inhibition of signaling by a conformation-sensitive GHR ECD monoclonal antibody. Further, the chimeras were much less sensitive to inducible metalloprotease cleavage than was WT GHR, implying that the ECD conformations of the chimera receptors differ from WT GHR. Collectively, our data indicate that the composition and/or length of the TMD affect some aspects of GHR function, but do not affect receptor predimerization or GH-induced GHR activation. Further, they suggest that the GHR ECD-TMD is more flexible than previously thought in terms of the ability to achieve the active conformation in response to GH.     

Regulation of full-length and truncated growth hormone (GH) receptor by GH in tissues of lit/lit or bovine GH transgenic mice

Two truncated isoforms of growth hormone (GH) receptor (GHR) were identified in mice and in humans. The proteins encoded by these isoforms lack most of the intracellular domain of the GHR and inhibit GH action in a dominant negative fashion. We have quantified the mRNAs encoding the GHR isoforms in mouse tissues by use of real-time RT-PCR and examined the effect of GH excess or deficiency on regulation of mRNA levels of the GHR isoforms in vivo. In the liver, the truncated GHR mRNAs (mGHR-282 and mGHR-280) were 0.5 and <0.1%, respectively, the level of full-length GHR (mGHR-fl). In skeletal muscle, the values were 2-3 and 0.1-0.5% of mGHR-fl, respectively, and in subcutaneous fat, the values were 3-5 and 0.1-0.5% of mGHR-fl, respectively. The bovine GH transgenic mice showed a significant increase of mGHR-fl in liver but a significant decrease in skeletal muscle, with no difference in subcutaneous fat when compared with control mice. The lit/lit mice showed a significant decrease of mGHR-fl in liver, no difference of mGHR-fl in muscle, and a significant increase of mGHR-fl in subcutaneous fat when compared with lit/+ mice. The mRNA of mGHR-282 was regulated in parallel with mGHR-fl in all tissues of all mice examined, whereas that of mGHR-280 was not changed in either GH-excess or GH-deficient states. In conclusion, two truncated isoforms of GHR mRNAs were detected in liver, skeletal muscle, and subcutaneous fat of mice. The ratio of GHR-tr to GHR-fl mRNA was tissue specific and not affected by chronic excess or deficiency of GH.     

Growth hormone (GH) receptor knockout mice reveal actions of GH in lung development

The presence of growth hormone (GH) and GH receptors (GHRs) in the lung suggests it is an autocrine/paracrine target site for pulmonary GH action and/or an endocrine site of pituitary GH action. Roles for GH in lung growth or pulmonary function are, however, uncertain. The possibility that pituitary and/or pulmonary GH have physiological roles in lung development has therefore been investigated in GHR knockout (KO or -/-) mice, using a proteomics approach to determine if an absence of GH-signaling affects the proteome of the developing lung. More than 600 proteins were detected by 2-DE in the lungs of control [GHR (+/+)] and GHR (-/-) mice at the end of the alveolarization period (at day 14 postnatally). Of these, 39 differed significantly in protein content at the p>0.05 level [6 were of higher abundance in the GHR (-/-) group, 33 were of lower abundance] and 17 differed at the p>0.02 level [5 of higher abundance in the GHR (-/-) group, 12 of lower abundance] and 7 were definitively identified by MS. Vimentin, a protein involved in cellular proliferation, was reduced in content by approximately 75% in the lungs of the GHR (-/-) mice. Three proteins involved in oxidative protection [SH3 domain-binding glutamic acid-rich-like protein, peroxiredoxin 6 (Prdx6), and isocitrate dehydrogenase 1] were also of lower content in the GHR (-/-) lungs (by approximately 88%, 81% and 70%, respectively). Prdx6 is also involved in lipid and surfactant metabolism, as is apolipoprotein A-IV, the lung content of which was reduced by approximately 73% in these mice. Proteasome 26S ATPase subunit 4, a protein involved in the non-lysosomal degradation of intracellular proteins, and electron flavoprotein alpha subunit , involved in intracellular metabolism, were also reduced in content in the lungs of the GHR (-/-) mice (by approximately 70% and 49%, respectively). These results therefore suggest that these proteins are normally dependent upon GH signaling, and that GH is normally involved in early lung growth, oxidative protection, lipid and energy metabolism and in proteasomal activity. These roles may reflect endocrine actions of pituitary GH and/or local autocrine/paracrine actions of GH produced within the lung.     

γ-Secretase-mediated growth hormone receptor proteolysis: mapping of the intramembranous cleavage site

GH receptor (GHR) undergoes regulated proteolysis by both metalloprotease (α-secretase) and γ-secretase activities. α-Secretase activity regulates GHR availability and sensitivity and generates circulating GH binding protein. The function of γ-secretase cleavage is yet uncertain. We investigated GHR determinants that affect inducible sequential α- and γ-secretase cleavage and thus remnant and stub generation, respectively. Purification and N-terminal sequencing of the stub revealed that γ-secretase cleavage occurs at an ε-site in GHR’s transmembrane domain four residues from the intracellular domain. Mutagenesis revealed that deletion of the proximal two transmembrane residues prevented both α- and γ-secretase-mediated proteolysis and deletion of four residues around the ε-site precluded surface GHR expression and proteolysis. However, point mutations in and around the ε-site affected neither α- or γ-secretase cleavage. We conclude that both cleavages likely occur at the cell surface and sequentially (α-secretase followed by γ-secretase) and that ε-site cleavage by γ-secretase does not require a consensus sequence.     

Growth hormone receptor is a target for presenilin-dependent gamma-secretase cleavage

Growth hormone receptor (GHR) is a cytokine receptor superfamily member that binds growth hormone (GH) via its extracellular domain and signals via interaction of its cytoplasmic domain with JAK2 and other signaling molecules. GHR is a target for inducible metalloprotease-mediated cleavage in its perimembranous extracellular domain, a process that liberates the extracellular domain as the soluble GH-binding protein and leaves behind a cell-associated GHR remnant protein containing the transmembrane and cytoplasmic domains. GHR metalloproteolysis can be catalyzed by tumor necrosis factor-alpha-converting enzyme (ADAM-17) and is associated with down-modulation of GH signaling. We now study the fate of the GHR remnant protein. By anti-GHR cytoplasmic domain immunoblotting, we observed that the remnant induced in response to phorbol ester or platelet-derived growth factor has a reliable pattern of appearance and disappearance in both mouse preadipocytes endogenously expressing GHR and transfected fibroblasts expressing rabbit GHR. Lactacystin, a specific proteasome inhibitor, did not appreciably change the time course of remnant appearance or clearance but allowed detection of the GHR stub, a receptor fragment slightly smaller than the remnant but containing the C terminus of the remnant (receptor cytoplasmic domain). In contrast, MG132, another (less specific) proteasome inhibitor, strongly inhibited remnant clearance and prevented stub appearance. Inhibitors of gamma-secretase, an aspartyl protease, also prevented the appearance of the stub, even in the presence of lactacystin, and concomitantly inhibited remnant clearance in the same fashion as MG132. In addition, mouse embryonic fibroblasts derived from presenilin 1 and 2 (PS1/2) knockouts recapitulated the gamma-secretase inhibitor studies, as compared with their littermate controls (PS1/2 wild type). Confocal microscopy indicated that the GHR cytoplasmic domain became localized to the nucleus in a fashion dependent on PS1/2 activity. These data indicate that the GHR is subject to sequential proteolysis by metalloprotease and gamma-secretase activities and may suggest GH-independent roles for the GHR.     

SCF acts in endosomal sorting of the GH receptor

The ubiquitin ligase SCF^TrCP is required for internalisation of the growth hormone receptor (GHR) and acts via a direct interaction with the ubiquitin-dependent endocytosis motif. Details of how the ligase communicates its information to the clathrin-mediated internalisation machinery are unknown. For the EGF receptor, c-Cbl acts both at the cell surface and in endosomes. We hypothesised that SCF^TrCP is required for GHR degradation at both sites. This was tested by truncating GHR after a di-leucine-based internalisation motif (GHR349). This receptor enters the cells via the adapter complex AP2. We show that TrCP acts in an early stage of cargo selection: both TrCP silencing and mutation of the ubiquitin-dependent endocytosis motif force the GHR to recycle between endosomes and the plasma membrane, together with the transferrin receptor. Depletion of Tsg101 (ESCRT-I) has the same effect, while silencing of Hrs (ESCRT-0) prevents GH recycling. GH passes through late endosomal vesicles, marked by Lamp1. Coexpressing GHR and EGFR demonstrates that both receptors use the same route to the lysosomes. We show for the first time that SCF^TrCP is involved in cargo-specific sorting at endosomes and that Tsg101 rather than Hrs might direct the cargo into the ESCRT machinery.     

A single amino acid substitution in the exoplasmic domain of the human growth hormone (GH) receptor confers familial GH resistance (Laron syndrome) with positive GH-binding activity by abolishing receptor homodimerization.

Growth hormone (GH) elicits a variety of biological activities mainly mediated by the GH receptor (GHR), a transmembrane protein that, based on in vitro studies, seemed to function as a homodimer. To test this hypothesis directly, we investigated patients displaying the classic features of Laron syndrome (familial GH resistance characterized by severe dwarfism and metabolic dysfunction), except for the presence of normal binding activity of the plasma GH-binding protein, a molecule that derives from the exoplasmic-coding domain of the GHR gene. In two unrelated families, the same GHR mutation was identified, resulting in the substitution of a highly conserved aspartate residue by histidine at position 152 (D152H) of the exoplasmic domain, within the postulated interface sequence involved in homodimerization. The recombinant mutated receptor protein was correctly expressed at the plasma membrane. It displayed subnormal GH-binding activity, a finding in agreement with the X-ray crystal structure data inferring this aspartate residue outside the GH-binding domain. However, mAb-based studies suggested the critical role of aspartate 152 in the proper folding of the interface area. We show that a recombinant soluble form of the mutant receptor is unable to dimerize, the D152H substitution also preventing the formation of heterodimers of wild-type and mutant molecules. These results provide in vivo evidence that monomeric receptors are inactive and that receptor dimerization is involved in the primary signalling of the GH-associated growth-promoting and metabolic actions.     

SCF(TrCP) acts in endosomal sorting of the GH receptor

The ubiquitin ligase SCF(TrCP) is required for internalisation of the growth hormone receptor (GHR) and acts via a direct interaction with the ubiquitin-dependent endocytosis motif. Details of how the ligase communicates its information to the clathrin-mediated internalisation machinery are unknown. For the EGF receptor, c-Cbl acts both at the cell surface and in endosomes. We hypothesised that SCF(TrCP) is required for GHR degradation at both sites. This was tested by truncating GHR after a di-leucine-based internalisation motif (GHR349). This receptor enters the cells via the adapter complex AP2. We show that TrCP acts in an early stage of cargo selection: both TrCP silencing and mutation of the ubiquitin-dependent endocytosis motif force the GHR to recycle between endosomes and the plasma membrane, together with the transferrin receptor. Depletion of Tsg101 (ESCRT-I) has the same effect, while silencing of Hrs (ESCRT-0) prevents GH recycling. GH passes through late endosomal vesicles, marked by Lamp1. Coexpressing GHR and EGFR demonstrates that both receptors use the same route to the lysosomes. We show for the first time that SCF(TrCP) is involved in cargo-specific sorting at endosomes and that Tsg101 rather than Hrs might direct the cargo into the ESCRT machinery.     

IGF-I deficiency due to GH receptor deficiency.

IGF-I deficiency may be primary due to defective synthesis, or secondary to GH receptor deficiency (GHRD) or defects in transduction of the GH-GHR signal. Cloning and sequencing of the GHR led to recognition that circulating GH binding protein (GHBP) was structurally identical to the extra-cellular domain of the GHR, and the identification of 33 mutations of the GHR in approximately half of the 250 patients that have been reported. This review explores the information provided about GHR function by various mutations, the population distribution of GHRD, the effects of this condition on mortality, growth, development, and metabolism, the effects of replacement therapy with recombinant human IGF-I, diagnostic issues, and the question of partial GH resistance.     

Identification of a region critical for proteolysis of the human growth hormone receptor.

Release of soluble growth hormone binding protein (GHBP) corresponding to the extracellular domain of the GH receptor (GHR) occurs via distinct mechanisms depending on species. In human, proteolysis of full length GHR results in liberation of GHBP into the extracellular medium. A putative protease responsive for GHR cleavage has been identified, however, the residues involved are still unknown. In this study, using the mutational approach to the extracellular domain of the human GHR, we demonstrated that deletion of three residues located close to the transmembrane domain abolishes constitutive GHBP shedding without change in cellular GH binding. Deletion also significantly decreased the phorbol 12-myristate 13-acetate (PMA)-induced release of GHBP and the accumulation of membrane-anchored remnant proteins. Taken together, these results suggest that integrity of the juxtamembrane region of GHR is necessary for its biochemical cleavage and that a common mechanism is involved in constitutive and PMA-induced shedding.