BLOCKADE OF GROWTH-HORMONE RECEPTOR SHEDDING BY A METALLOPROTEASE INHIBITOR

GH, an important growth-promoting and metabolic hormone, exerts its bi ological effects by interacting with cell surface GH receptors (GHRs). The GHR is a single membrane-spanning protein that binds GH via its e xtracellular domain. The high affinity GH-binding protein (GHBP), whic h corresponds to a soluble form of the GHR extracellular domain, carri es a substantial fraction of the GH in the circulation of various spec ies and probably has a role in modulation of the hormone's bioavailabi lity. Although in rodents, it is believed that the GHBP is largely der ived by translation of an alternatively spliced GHR messenger RNA, in humans and rabbits, proteolytic cleavage of the membrane-anchored rece ptor releases the GHR extracellular domain, which is believed to there by become the GHBP. In this study, we used human IM-9 lymphocytes and GHR antibodies to study this proteolytic shedding of the GHBP. As dete rmined by immunoblotting with anti-GHR cytoplasmic domain serum, addit ion of phorbol 12-myristate 13-acetate (PMA; 1 mu g/ml) to serum-starv ed cells led to rapid loss (roughly 60% decline after 1 h; t(1/2) = si milar to 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 c ytoplasmically 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 progres sively faster migrating forms were evident between 5-60 min of PMA exp osure. 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 block ed by the protein kinase C inhibitor, GF109203X. Both PMA- and NEM-ind uced receptor proteolysis were, however, inhibited by the metalloprote ase inhibitor, Immunex Compound 3 (minimum effective concentration, 10 mu M). Notably, PMA and NEM also promoted shedding of GHBP into the c onditioned medium of the cells, as determined by a chromatographic [I- 125]human GH binding assay; this GHBP shedding was also inhibited by I mmunex Compound 3. These results strongly implicate a member(s) of the metalloprotease family as a potential GHBP-generating enzyme.