Phorbol ester- and growth factor-induced growth hormone (GH) receptor proteolysis and GH-Binding protein shedding: Relationship to GH receptor down-regulation

GH signals by interacting with GH receptor (GHR). A substantial fraction of circulating GH complexes with GH-binding protein (GHBP), which corresponds to the GHR extracellular domain. GHBP is generated by 1) alternative splic ing of a common GHR precursor messenger RNA to encode secreted GHBP (the so urce of the vast majority of GHBP in rodents); and 2) proteolysis of the ce ll-associated GHR with shedding of GHBP (a mechanism operative in rabbits a nd humans). We previously observed that phorbol eater (PMA)-induced activat ion of protein kinase C (PKC) causes metalloprotease-mediated GHR proteolys is and GHBP shedding in human IM-9 lymphocytes. We now demonstrate that PMA -induced hydroxamate (IC3)-inhibitable GHR proteolysis and GHBP shedding we re also detected in murine 3T3-F442A and 3T3-L1 preadipocytes and in Chines e hamster ovary (CHO) cells stably expressing rabbit GHR (rbGHR), although the degree of GHBP shedding was much smaller for murine GHR than for rabbit or human GHRs. PMA-induced GHR proteolysis in 3T3-F442A, 3T3-L1, and CHO-r bGHR cells was significantly reduced by pretreatment with mitogen-activated protein kinase/extra-cellular signal-regulated kinase kinase 1 inhibitors, suggesting involvement of the mitogen-activated protein kinase pathway in regulating this PKC-dependent effect. In contrast, GHR proteolysis promoted by N-ethylmaleimide, although inhibited by IC3, was unaffected by inhibiti on of either PKC or mitogen-activated protein kinase/extracellular signal-r egulated kinase kinase 1. Thus, different pathways leading to metalloprotea se-mediated receptor proteolysis are accessed by PMA vs. N-ethylmaleimide. To determine whether other, possibly more physiologically relevant, stimuli induce GHR proteolysis, we tested effects of platelet-derived growth facto r (PDGF) and serum. Treatment of serum-deprived cells with PDGF tin 3T3-F44 2A cells) or serum (in 3T3-F442A and CHO-rbGHR cells) promoted GHR proteoly sis, which was inhibited by IC3. interestingly, PMA-, PDGF-, and serum-indu ced GHR proteolysis was associated with substantial decreases in GH-induced activation of janus kinase-a, which were also prevented by IC3. These find ings suggest that inducible metalloprotease-mediated GHR proteolysis consti tutes an important mechanism of receptor down-regulation and modulation of GH signaling.