Blockade of Growth Hormone Receptor Shedding by a Metalloprotease Inhibitor1

GH, an important growth-promoting and metabolic hormone, exerts its biological effects by interacting with cell surface GH receptors (GHRs). The GHR is a single membrane-spanning protein that binds GH via its extracellular domain. The high affinity GH-binding protein (GHBP), which corresponds to a soluble form of the GHR extracellular domain, carries a substantial fraction of the GH in the circulation of various species and probably has a role in modulation of the hormone’s bioavailability. Although in rodents, it is believed that the GHBP is largely derived by translation of an alternatively spliced GHR messenger RNA, in humans and rabbits, proteolytic cleavage of the membrane-anchored receptor releases the GHR extracellular domain, which is believed to thereby become the GHBP. In this study, we used human IM-9 lymphocytes and GHR antibodies to study this proteolytic shedding of the GHBP. As determined by immunoblotting with anti-GHR cytoplasmic domain serum, addition of phorbol 12-myristate 13-acetate (PMA; 1μ g/ml) to serum-starved cells led to rapid loss (roughly 60% decline after 1 h; t1/2 = ∼5 min) of mature GHRs (115–140 kDa) from either total cell or detergent-soluble extracts. Loss of full-length GHRs was accompanied by accumulation of four proteins (65–68 kDa), each reactive with the cytoplasmically directed antiserum. The pattern of appearance of these GHR ctyoplasmic domain proteins, the electrophoretic and immunological characteristics of which are similar to those of a recombinant rabbit GHR mutant that lacks the extracellular domain, was such that progressively faster migrating forms were evident between 5–60 min of PMA exposure. Treatment with N-ethylmaleimide (NEM; 5 mm), an agent known to cause GHBP shedding from IM-9 cells, promoted a similar rapid loss of full-length GHRs and an accumulation of GHR cytoplasmic domain remnant proteins. PMA-induced, but not NEM-induced, GHR proteolysis was blocked by the protein kinase C inhibitor, GF109203X. Both PMA- and NEM-induced receptor proteolysis were, however, inhibited by the metalloprotease inhibitor, Immunex Compound 3 (minimum effective concentration, 10 μm). Notably, PMA and NEM also promoted shedding of GHBP into the conditioned medium of the cells, as determined by a chromatographic [125I]human GH binding assay; this GHBP shedding was also inhibited by Immunex Compound 3. These results strongly implicate a member(s) of the metalloprotease family as a potential GHBP-generating enzyme.