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.

Janus kinase 2 influences growth hormone receptor metalloproteolysis.

GH signals through the GH receptor (GHR), a cytokine receptor superfamily member that couples to the cytoplasmic tyrosine kinase, Janus kinase 2 (JAK2). In addition to its role in signaling, we recently implicated JAK2 in the regulation of cell surface GHR abundance by modulation of GHR trafficking and mature GHR stability. GHR is a target for constitutive and inducible metalloprotease-mediated cleavage that alters surface GHR levels and can modulate GH signaling. We previously found that metalloprotease cleavage of GHR is dramatically lessened in fibroblasts derived from mice with targeted deletion of the zinc-binding domain of TNF-alpha-cleaving enzyme [TACE; ADAM17 (a disintegrin and metalloprotease)], implicating this transmembrane ectoenzyme as a GHR metalloprotease. In this study we used a human fibrosarcoma reconstitution system to compare the effects of RNA interference-mediated knockdown of TACE vs. a related metalloprotease, ADAM10. We found that TACE knockdown dramatically reduced both the pace and the degree of inducible GHR proteolysis and augmented the abundance of mature GHR, suggesting a role for TACE in constitutive receptor proteolysis in this system as well. Notably, ADAM10 knockdown also reduced inducible GHR proteolysis, although to a lesser degree than TACE knockdown, suggesting a contribution from this metalloprotease also. To determine whether JAK2 affects GHR proteolysis, we compared JAK2-deficient vs. JAK2-replete cells and found that phorbol 12-methyl 13-acetate-induced GHR proteolysis was significantly diminished in cells that lacked JAK2. Reconstitution with a GHR mutant that lacks the box 1 region (which mediates JAK2 association) resulted in phorbol 12-methyl 13-acetate-induced proteolysis similar in degree to that of the wild-type GHR in JAK2-deficient cells. Introduction of JAK2 did not affect the proteolysis of this box 1-deleted GHR, suggesting GHR-JAK2 association is required for JAK2 to affect GHR proteolysis. Additionally, the inhibitory effect of anti-GHRext-mAb, a conformation-sensitive GHR antibody, on receptor proteolysis was lost in cells that lacked JAK2. Our data indicate that the susceptibility of GHR to proteolysis is substantially affected by JAK2, suggesting yet another role for this kinase in determining GH sensitivity.

Janus kinase 2 enhances the stability of the mature growth hormone receptor.

The abundance of surface GH receptor (GHR) is an important determinant of cellular GH sensitivity and is regulated at both transcriptional and posttranscriptional levels. In previous studies of GHR-expressing Janus kinase 2 (JAK2)-deficient human fibrosarcoma cells (gamma2A-GHR), we demonstrated that stable transfection with JAK2 resulted in increased steady-state levels of mature GHR (endoH-resistant; relative molecular mass, 115-140 kDa) relative to precursor GHR (endoH-sensitive; relative molecular mass, 100 kDa). We now examine further the effects of JAK2 on GHR trafficking by comparing gamma2A-GHR to gamma2A-GHR cells stably reconstituted with JAK2 (C14 cells). In the presence of JAK2, GHR surface expression was increased, as assessed by surface biotinylation, 125I-labeled human GH cell surface binding, and immunofluorescence microscopy assays. Although the absence of JAK2 precluded GH-stimulated signaling, GH-induced GHR disulfide linkage (a proxy for the GH-induced conformational changes in the GHR dimer) proceeded independent of JAK2 expression, indicating that the earliest steps in GH-induced GHR triggering are not prevented by the absence of JAK2. RNA interference-mediated knockdown of JAK2 in C14 cells resulted in a decreased mature to precursor ratio, supporting a primary role for JAK2 either in enhancing GHR biogenesis or dampening mature GHR degradation. To address these potential mechanisms, metabolic pulse-chase labeling experiments and experiments in which the fate of previously synthesized GHR was followed by anti-GHR immunoblotting after cycloheximide treatment (cycloheximide chase experiments) were performed. These indicated that the presence of JAK2 conferred modest enhancement (1.3- to 1.5-fold) in GHR maturation but substantially prolonged the t1/2 of the mature GHR, suggesting a predominant effect on mature GHR stability. Cycloheximide chase experiments with metalloprotease, proteasome, and lysosome inhibitors indicated that the enhanced stability of mature GHR conferred by JAK2 is not related to effects on constitutive receptor metalloproteolysis but rather is a result of reduced constitutive endosomal/lysosomal degradation of the mature GHR. These results are discussed in the context of emerging information on how JAK-family members modulate surface expression of other cytokine receptors.

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.