Constitutive Proteolysis of the ErbB-4 Receptor Tyrosine Kinase by a Unique, Sequential Mechanism

The heregulin receptor tyrosine kinase ErbB-4 is constitutively cleaved, in the presence or absence of ligand, by an exofacial proteolytic activity producing a membrane-anchored cytoplasmic domain fragment of 80 kD. Based on selective sensitivity to inhibitors, the proteolytic activity is identified as that of a metalloprotease. The 80-kD product is tyrosine phosphorylated and retains tyrosine kinase activity. Importantly, the levels of this fragment are controlled by proteasome function. When proteasome activity is inhibited for 6 h, the kinase-active 80-kD ErbB-4 fragment accumulates to a level equivalent to 60% of the initial amount of native ErbB-4 (~10⁶ receptors per cell). Hence, proteasome activity is essential to prevent the accumulation of a significant level of ligand-independent, active ErbB-4 tyrosine kinase generated by metalloprotease activity. Proteasome activity, however, does not act on the native ErbB-4 receptor before the metalloprotease-mediated cleavage, as no ErbB-4 fragments accumulate when metalloprotease activity is blocked. Although no ubiquitination of the native ErbB-4 is detected, the 80-kD fragment is polyubiquitinated. The data, therefore, describe a unique pathway for the processing of growth factor receptors, which involves the sequential function of an exofacial metalloprotease and the cytoplasmic proteasome.     

Role of the ErbB-4 carboxyl terminus in gamma-secretase cleavage

The ErbB-4 receptor tyrosine kinase has a PDZ domain recognition motif at its carboxyl terminus. The first step in ErbB-4 proteolytic processing is a metalloprotease-dependent cleavage of the receptor ectodomain, which is not influenced by deletion of this motif. Metalloprotease cleavage of ErbB-4 produces a membrane-associated 80-kDa fragment that is a substrate for subsequent gamma-secretase cleavage, which releases the cytoplasmic domain from the membrane and allows nuclear translocation of this fragment. Deletion of the PDZ domain recognition motif does abrogate the gamma-secretase cleavage of ErbB-4. The wild-type 80-kDa ErbB-4 fragment forms an association complex with presenilin, thought to be the catalytic moiety of gamma-secretase activity. However, this association is significantly impaired by loss of the PDZ domain recognition motif from ErbB-4. Deletion of this ErbB-4 motif prevents the nuclear localization of the ErbB-4 cytoplasmic domain. Data also show that the basal cleavage of wild-type ErbB-4 by this proteolytic system can produce a sufficient level of ErbB-4 processing to negatively influence cell growth and that loss of the PDZ domain recognition motif abrogates this response.     

Geldanamycin induces ErbB-2 degradation by proteolytic fragmentation

Exposure of carcinoma cell lines to the antibiotic geldanamycin induces the degradation of ErbB-2, a co-receptor tyrosine kinase that is frequently overexpressed in certain tumors. Using ErbB-2 mutants expressed as chimeric receptors or green fluorescent protein fusion proteins, we report that the kinase domain of ErbB-2 is essential for geldanamycin-induced degradation. The kinase domain of the related epidermal growth factor receptor was not sensitive to this drug. The data further indicate mechanistic aspects of ErbB-2 degradation by geldanamycin. The data show that exposure to the drug induces at least one cleavage within the cytoplasmic domain of ErbB-2 producing a 135-kDa fragment and a 23-kDa fragment. The latter represents the carboxyl-terminal domain of ErbB-2, whereas the former represents the ectodomain and part of the cytoplasmic domain. Degradation of the carboxyl-terminal fragment is prevented by proteasome inhibitors, whereas degradation of the membrane-anchored 135-kDa ErbB-2 fragment is blocked by inhibitors of the endocytosis-dependent degradation pathway. Confocal microscopy studies confirm a geldanamycin-induced localization of ErbB-2 on intracellular vesicles.     

Ectodomain cleavage of ErbB-4: characterization of the cleavage site and m80 fragment

Ectodomain cleavage of the ErbB-4 receptor tyrosine kinase generates a membrane-associated fragment of 80 kDa (m80) that has been subjected to N-terminal sequencing. The sequence obtained shows that the N terminus of this fragment begins with Ser-652 of ErbB-4. When a 12-residue peptide corresponding to ErbB-4 residues 646-657 was incubated with recombinant tumor necrosis factor-alpha-converting enzyme, fragments representing residues 646-651 and 652-657 were obtained. These data indicate that ectodomain cleavage of ErbB-4 occurs between His-651 and Ser-652, placing the cleavage site within the ectodomain stalk region approximately 8 residues prior to the transmembrane domain. Several experiments have characterized other aspects of the m80 ErbB-4 fragment. Inhibition of ErbB-4 tyrosine kinase activity with pan-ErbB tyrosine kinase inhibitors indicates that kinase activity is stringently required for heregulin-dependent, but not 12-O-tetradecanoylphorbol-13-acetate-induced, ErbB-4 ectodomain cleavage and formation of the m80 fragment. When the m80 ErbB-4 fragment is generated by cell treatment with heregulin or 12-O-tetradecanoylphorbol-13-acetate, the fragment associates with intact ErbB-2. However, this fragment does not associate with the intact ErbB-4 molecule.     

Caspase-dependent cleavage of ErbB-2 by geldanamycin and staurosporin

The geldanamycin-induced degradation of ErbB-2 produces a 23-kDa carboxyl-terminal fragment, which has been isolated and subjected to amino-terminal microsequencing. The obtained sequence indicates that the amino terminus of this fragment corresponds to Gly-1126 of ErbB-2. Analysis of the residues immediately before Gly-1126 suggests that cleavage may involve caspase activity. Site-directed mutagenesis of Asp-1125 in ErbB-2 prevents geldanamycin-provoked formation of the 23-kDa fragment, consistent with the requirement of this residue for caspase-dependent cleavage in known substrates. Also, the addition of the pan-caspase inhibitor Z-VAD-FMK blocks formation of the 23-kDa ErbB-2 fragment in cells exposed to geldanamycin. Interestingly, staurosporin and curcumin are also shown to provoke the degradation of ErbB-2 with formation of the 23-kDa carboxyl-terminal fragment. The generation of this fragment by staurosporin or curcumin is likewise blocked by caspase inhibition. Caspase inhibition does not prevent accelerated degradation of the 185-kDa native ErbB-2 in geldanamycin-treated cells but does significantly prevent staurosporin-stimulated metabolic loss of ErbB-2.     

Matrix metalloproteinase-1 induces cleavage of exogenous alphaB-crystallin transduced by a cell-penetrating peptide

Cell-penetrating peptides (CPPs), including TAT-CPP, have been used to deliver exogenous proteins into living cells. Although a number of proteins fused to TAT-CPP can be delivered into various cells, little is known about the proteolytic cleavage of TAT-fusion proteins in cells. In this study, we demonstrate that a small heat shock protein (sHSP), alphaB-crystallin (αB-crystallin), delivered by TAT-CPP is susceptible to proteolytic cleavage by matrix metalloproteinase-1 (MMP-1) in cardiac myoblast H9c2 cells. Recombinant TAT-αB-crystallin was efficiently transduced into H9c2 cells. For a few hours following protein transduction, generation of a 14-kDa fragment, a cleavage band of TAT-αB-crystallin, increased in a time-dependent manner. This fragment was observed only in detergent-insoluble fractions. Interestingly, treatment with MMP inhibitors blocked the cleavage of TAT-αB-crystallin. In test tubes, recombinant MMP-1 processed TAT-αB-crystallin to generate the major cleavage fragment 14-kDa, as observed in the cells treated with TAT-αB-crystallin. The N-terminal sequences of the 14-kDa fragment were identified as Leu-Arg-Ala-Pro-Ser-Trp-Phe, indicating that this fragment is generated by cleavage at Phe54-Leu55 of αB-crystallin. The MMP-1-selective inhibitor abolished the production of 14-kDa fragments in cells. In addition, the cleaved fragment of TAT-αB-crystallin was significantly reduced in cells transfected with MMP-1 siRNA. Moreover, the enzymatic activity of MMP-1 was markedly increased in TAT-αB-crystallin-treated cells. TAT-αB-crystallin has a cytoprotective effect on H9c2 cells under hypoxic insult, moreover, MMP-1-selective inhibitor treatment led to even increased cell viability. These results suggest that MMP-1 is responsible for proteolytic cleavage of TAT-αB-crystallin during its intracellular transduction in H9c2 cells.     

ErbB-4 and TNF-alpha converting enzyme localization to membrane microdomains

Sequential proteolytic processing of ErbB-4 occurs in response to ligand addition. Here, we assess the localization of cleavable and non-cleavable ErbB-4 isoforms to membrane microdomains using three methodologies: (1) Triton X-100-insolubility, (2) Brij98-insolubility, and (3) detergent-free density gradient centrifugation. Whereas ErbB-4 translocated to a Triton X-100-insoluble fraction upon treatment of T47D cells with heregulin, it constitutively associated with a Brij98-insoluble fraction and a lipid raft fraction isolated using detergent-free methodology. Comparison of cleavable and non-cleavable isoforms of ErbB-4 revealed that both ErbB-4 isoforms are constitutively localized to either a Triton X-100-soluble or Brij98-insoluble fraction. In contrast, addition of heregulin resulted in translocation of the cleavable isoform to a detergent-free lipid raft. Tumor necrosis factor-alpha converting enzyme (TACE), the ectodomain secretase for ErbB-4, was present predominantly in its mature active form in most microdomains analyzed. These data suggest the assembly of ErbB-4 ectodomain cleavage apparatus in a membrane microdomain.     

Differential effects of proteases involved in intracellular degradation of amyloid beta-protein between detergent-soluble and -insoluble pools in CHO-695 cells.

The deposition of amyloid beta-protein (A beta or beta A4) is a key feature of Alzheimer's disease. Most studies have focused on the generation of A beta, but little is known about the degradation of A beta. Recent reports suggest that insulin-degrading enzyme (IDE) and neutral endopeptidase (NEP) are involved in the extracellular degradation of A beta. To date, however, far less is known about the degradation of intracellular A beta. To elucidate the protease(s) responsible for the degradation of intracellular A beta, we investigated the effect of various protease inhibitors on A beta in two distinct intracellular pools (i.e., nonionic detergent-soluble and detergent-insoluble pools) in Chinese hamster ovary cells. Treatment with thiol and metal inhibitors resulted in the accumulation of intracellular A beta and oligomers in detergent-soluble and -insoluble fractions. The overexpression of thiol-metalloprotease IDE resulted in a marked reduction in levels of detergent-soluble intracellular A beta as well as extracellular A beta 40 and A beta 42. Moreover, intracellular A beta in the detergent-insoluble fraction extracted with 70% formic acid or 6 M guanidine hydrochloride decreased markedly in the cells overexpressing IDE. In contrast, expression of NEP degraded the A beta in the detergent-insoluble fraction markedly and partially degraded extracellular A beta 40 and A beta 42, but not intracellular soluble A beta. Thiorphan, an inhibitor of NEP, accumulated, albeit to a lesser extent, in insoluble A beta but not in soluble A beta. Thus, IDE appears to degrade intracellular A beta more effectively than does NEP in both the detergent-soluble and -insoluble fractions.     

Betacellulin-induced beta cell proliferation and regeneration is mediated by activation of ErbB-1 and ErbB-2 receptors

Background

Betacellulin (BTC), a member of the epidermal growth factor family, is known to play an important role in regulating growth and differentiation of pancreatic beta cells. Growth-promoting actions of BTC are mediated by epidermal growth factor receptors (ErbBs), namely ErbB-1, ErbB-2, ErbB-3 and ErbB-4; however, the exact mechanism for beta cell proliferation has not been elucidated. Therefore, we investigated which ErbBs are involved and some molecular mechanisms by which BTC regulates beta cell proliferation.

Methodology/Principal Findings

The expression of ErbB-1, ErbB-2, ErbB-3, and ErbB-4 mRNA was detected by RT-PCR in both a beta cell line (MIN-6 cells) and C57BL/6 mouse islets. Immunoprecipitation and western blotting analysis showed that BTC treatment of MIN-6 cells induced phosphorylation of only ErbB-1 and ErbB-2 among the four EGF receptors. BTC treatment resulted in DNA synthetic activity, cell cycle progression, and bromodeoxyuridine (BrdU)-positive staining. The proliferative effect was blocked by treatment with AG1478 or AG825, specific tyrosine kinase inhibitors of ErbB-1 and ErbB-2, respectively. BTC treatment increased mRNA and protein levels of insulin receptor substrate-2 (IRS-2), and this was blocked by the ErbB-1 and ErbB-2 inhibitors. Inhibition of IRS-2 by siRNA blocked cell cycle progression induced by BTC treatment. Streptozotocin-induced diabetic mice injected with a recombinant adenovirus expressing BTC and treated with AG1478 or AG825 showed reduced islet size, reduced numbers of BrdU-positive cells in the islets, and did not attain BTC-mediated remission of diabetes.

Conclusions/Significance

These results suggest that BTC exerts proliferative activity on beta cells through the activation of ErbB-1 and ErbB-2 receptors, which may increase IRS-2 expression, contributing to the regeneration of beta cells.     

Proteasomes mediate prolactin-induced receptor down-regulation and fragment generation in breast cancer cells

Prolactin regulates a variety of physiological processes, including mammary gland growth and differentiation, and recent findings support an important role in breast cancer development and progression. However, little is known about the trafficking of its receptor, a member of the cytokine receptor superfamily. In the present study, we examined the effect of ligand on the endogenous "long" isoform of the prolactin receptor in breast cancer cells. We found that prolactin caused rapid and prolonged down-regulation of this receptor. The prolactin-induced increase in degradation was blocked by inhibitors of both proteasomes and lysosomes. However, the ubiquitin-conjugating system was not required for internalization. Prolactin also resulted in the concomitant appearance of a cell-associated prolactin receptor fragment containing the extracellular domain. This latter process required proteasomal, but not metalloprotease, activity, distinguishing it from ectodomain "shedding" of other membrane receptors, which are secreted as binding proteins. The prolactin receptor fragment was labeled by surface biotinylation and independent of protein synthesis. Together, these data indicated that prolactin binding initiates limited proteasomal cleavage of its receptor, generating a cell-associated fragment containing the extracellular domain. Our findings described a new potential mediator of prolactin action and a novel mechanism whereby proteasomes modulate cellular processes.     

Ectodomain shedding of nectin-1alpha by SF/HGF and TPA in MDCK cells

Nectin is a Ca(2+)-independent immunoglobulin-like cell-cell adhesion molecule implicated in the organization of the junctional complex comprised of E-cadherin-based adherens junctions and claudin-based tight junctions in epithelial cells. Scatter factor (SF)/hepatocyte growth factor (HGF) and 12-O-tetradecanoylphorbol-13-acetate (TPA), a tumor-promoting phorbol ester, induce cell spreading, followed by cell-cell dissociation and cell scattering, in Madin-Darby canine kidney (MDCK) cells. We found here that SF/HGF and TPA induced proteolytic cleavage of nectin-1alpha in the ectodomain, resulting in generation of the 80-kDa extracellular fragment and the 33-kDa fragment composed of the transmembrane and cytoplasmic domains, in MDCK cells. This shedding of nectin-1alpha was inhibited by metalloprotease inhibitors. These results indicate that SF/HGF and TPA induce the ectodomain shedding of nectin-1alpha presumably by a metalloprotease, and have raised the possibility that this shedding is involved in the SF/HGF- and TPA-induced cell-cell dissociation.     

ErbB-4: mechanism of action and biology

The most recently described member of the ErbB receptor tyrosine kinase family is ErbB-4. In general, the structure of this receptor and its mechanism of action is similar to that described for ErbB-1. However, significantly less is known about ErbB-4 and there are several novel aspects to its structure, mechanism of action, and biology. This includes the spectrum of ligands that activate ErbB-4, the presence of functionally distinct isoforms, a proteolytic processing pathway to the nucleus, and the capacity to induce a spectrum of cellular responses such as mitogenesis, differentiation, growth inhibition, and survival.     

The extracellular N terminus of the endothelin B (ETB) receptor is cleaved by a metalloprotease in an agonist-dependent process

The extracellular N terminus of the endothelin B (ET(B)) receptor is susceptible to limited proteolysis (cleavage at R64 downward arrow S65), but the regulation and the functional consequences of the proteolysis remain elusive. We analyzed the ET(B) receptor or an ET(B)-GFP fusion protein stably or transiently expressed in HEK293 cells. After incubation of cells at 4 degrees C, only the full-length ET(B) receptor was detected at the cell surface. However, when cells were incubated at 37 degrees C, N-terminal cleavage was observed, provided endothelin 1 was present during the incubation. Cleavage was not inhibited by internalization inhibitors (sucrose, phenylarsine oxide). However, in cells incubated with both internalization inhibitors and metalloprotease inhibitors (batimastat, inhibitor of TNFalpha-convertase) or metal chelators (EDTA, phenanthroline), the cleavage was blocked, indicating that metalloproteases cleave the agonist-occupied ET(B) receptor at the cell surface. Functional analysis of a mutant ET(B) receptor lacking the first 64 amino acids ([Delta2-64]ET(B) receptor) revealed normal functional properties, but a 15-fold reduced cell surface expression. The results suggest a role of the N-terminal proteolysis in the regulation of cell surface expression of the ET(B) receptor. This is the first example of a multispanning membrane protein, which is cleaved by a metalloprotease, but retains its functional activity and overall structure.     

ADAM10 mediates ectodomain shedding of the betacellulin precursor activated by p-aminophenylmercuric acetate and extracellular calcium influx

Betacellulin belongs to the family of epidermal growth factor-like growth factors that are expressed as transmembrane precursors and undergo proteolytic ectodomain shedding to release a soluble mature growth factor. In this study, we investigated the ectodomain shedding of the betacellulin precursor (pro-BTC) in conditionally immortalized wild-type (WT) and ADAM-deficient cell lines. Sequential ectodomain cleavage of the predominant cell-surface 40-kDa form of pro-BTC generated a major (26-28 kDa) and two minor (20 and 15 kDa) soluble forms and a cellular remnant lacking the ectodomain (12 kDa). Pro-BTC shedding was activated by calcium ionophore (A23187) and by the metalloprotease activator p-aminophenylmercuric acetate (APMA), but not by phorbol esters. Culturing cells in calcium-free medium or with the protein kinase Cdelta inhibitor rottlerin, but not with broad-based protein kinase C inhibitors, blocked A23187-activated pro-BTC shedding. These same treatments were without effect for constitutive and APMA-induced cleavage events. All pro-BTC shedding was blocked by treatment with a broad-spectrum metalloprotease inhibitor (GM6001). In addition, constitutive and activated pro-BTC shedding was differentially blocked by TIMP-1 or TIMP-3, but was insensitive to treatment with TIMP-2. Pro-BTC shedding was functional in cells from ADAM17- and ADAM9-deficient mice and in cells overexpressing WT or catalytically inactive ADAM17. In contrast, overexpression of WT ADAM10 enhanced constitutive and activated shedding of pro-BTC, whereas overexpression of catalytically inactive ADAM10 reduced shedding. These results demonstrate, for the first time, activated pro-BTC shedding in response to extracellular calcium influx and APMA and provide evidence that ADAM10 mediates constitutive and activated pro-BTC shedding.     

A Disintegrin and Metalloproteinase-10 (ADAM-10) Mediates DN30 Antibody-induced Shedding of the Met Surface Receptor

Met, the tyrosine kinase receptor for the hepatocyte growth factor is a prominent regulator of cancer cell invasiveness and has emerged as a promising therapeutic target. Binding of the anti-Met monoclonal antibody DN30 to its epitope induces the proteolytic cleavage of Met, thereby impairing the invasive growth of tumors. The molecular mechanism controlling this therapeutic shedding process has so far been unknown. Here, we report that A Disintegrin And Metalloproteinase (ADAM)-10, but not ADAM-17, is required for DN30-induced Met shedding. Knockdown of ADAM-10 in different tumor cell lines or abrogation of its proteolytic activity by natural or synthetic inhibitors abolished Met down-regulation on the cell surface as well as reduction of Met activation. Moreover, hepatocyte growth factor-induced tumor cell migration and invasion were impaired upon ADAM-10 knockdown. Thus, the therapeutic effect of DN30 involves ADAM-10-dependent Met shedding, linking for the first time a specific metalloprotease to target therapy against a receptor tyrosine kinase.     

Inhibition of the proteolytic activity of pregnancy-associated plasma protein-A by targeting substrate exosite binding

The metalloproteinase pregnancy-associated plasma protein-A (PAPP-A) cleaves both insulin-like growth factor (IGF)-binding protein 4 (IGFBP-4) and -5 at a single site in their central domain causing the release of bioactive IGF. Inhibition of IGF signaling is relevant in human disease, and several drugs in development target the IGF receptor. However, inhibition of PAPP-A activity may be a valuable alternative. We have generated monoclonal phage-derived single chain fragment variable (scFv) antibodies which selectively inhibit the cleavage of IGFBP-4 by PAPP-A, relevant under conditions where cleavage of IGFBP-4 represents the final step in the delivery of IGF to the IGF receptor. None of the antibodies inhibited the homologous proteinase PAPP-A2, which allowed mapping of antibody binding by means of chimeras between PAPP-A and PAPP-A2 to the C-terminal Lin12-Notch repeat module, separated from the proteolytic domain by almost 1000 amino acids. Hence, the antibodies define a substrate binding exosite that can be targeted for the selective inhibition of PAPP-A proteolytic activity against IGFBP-4. In addition, we show that the Lin12-Notch repeat module reversibly binds a calcium ion and that bound calcium is required for antibody binding, providing a strategy for the further development of selective inhibitory compounds. To our knowledge these data represent the first example of differential inhibition of cleavage of natural proteinase substrates by exosite targeting. Generally, exosite inhibitors are less likely to affect the activity of related proteolytic enzymes with similar active site environments. In the case of PAPP-A, selective inhibition of IGFBP-4 cleavage by interference with exosite binding is a further advantage, as the activity against other known or unknown PAPP-A substrates, whose cleavage may not depend on binding to the same exosite, is not targeted.     

The proprotein convertase PC5A and a metalloprotease are involved in the proteolytic processing of the neural adhesion molecule L1

The transmembrane and multidomain neural adhesion molecule L1 plays important functional roles in the developing and adult nervous system. L1 is proteolytically processed at two distinct sites within the extracellular domain, leading to the generation of different fragments. In this report, we present evidence that the proprotein convertase PC5A is the protease that cleaves L1 in the third fibronectin type III domain, whereas the proprotein convertases furin, PC1, PC2, PACE4, and PC7 are not effective in cleaving L1. Analysis of mutations revealed Arg(845) to be the site of cleavage generating the N-terminal 140-kDa fragment. This fragment was present in the hippocampus, which expresses PC5A, but was not detectable in the cerebellum, which does not express PC5A. The 140-kDa L1 fragment was found to be tightly associated with the full-length 200-kDa L1 molecule. The complex dissociated from the membrane upon cleavage by a protease acting at a more membrane-proximal site of full-length L1. This proteolytic cleavage was inhibited by the metalloprotease inhibitor GM 6001 and enhanced by a calmodulin inhibitor. L1-dependent neurite outgrowth of cerebellar neurons was inhibited by GM 6001, suggesting that proteolytic processing of L1 by a metalloprotease is involved in neurite outgrowth.     

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.