Release of Growth Hormone Binding Protein from IM-9 Lymphocytes by Endopeptidase is Dependent on Sulfydryl Group Inactivation*

Abstract

The hydrophilic GH-binding protein of serum is a derivative of the GH receptor. Little is known how this GH binding protein is released from the receptor which is firmly anchored in the plasma membrane. The IM-9 lymphocytes provide a useful laboratory model for studying this process because they are richly endowed with GH receptors and, under special conditions, are able to shed these receptors during incubation. Incubation of IM-9 cells for 90 min at 30 C did not result in the appearance of significant [125I]hGH binding in conditioned medium as determined with an ultrogel AcA 44 minicolumn. When iodoacetamide, 20 mM, or N-ethylmaleimide, 5 mM, was added during incubation, the conditioned medium bound 20-35% of [125I]human(h)GH. p-Chloromercuriphenyl sulfonic acid was less effective in promoting shedding of GH-binding protein. In contrast, aprotinin, phenylmethylsulfonylfluoride (PMSF), bacitracin, leupeptin, pepstatin, phosphoramidon, or chloroquine did not promote release of GH binding protein and did not affect iodoacetamide-induced release. Release was not inhibited by the addition of serum lacking GH binding protein. GH binding protein release was markedly temperature sensitive and practically ceased at 4 C. GH binding protein incubated with [125I] hGH was cross-linked with disuccinimidyl suberate. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of dithiothreitol the complex migrated with an estimated molecular weight of 100,000 whereas [125I]hGH cross-linked to the membrane-bound GH receptor of the IM-9 cells migrated with an estimated molecular weight of 135,000. The smaller size of the binding protein is consistent with its derivation from the extracellular domain of the GH receptor. Because the release of this GH binding is greatly augmented by iodoacetamide and N-ethylmaleimide, two known sulfhydryl reactive reagents, we suggest that a free sulfhydryl group, either on the GH receptor or on a neighboring protein normally maintains the integrity of the receptor. The loss of this sulfhydryl group destabilizes the receptor and permits a membrane endopeptidase to release the GH binding protein. Cleavage is not dependent on lysosomal action and is not inhibited by protease inhibitors.

Shedding of growth hormone-binding protein is inhibited by hydroxamic acid-based protease inhibitors: proposed mechanism of activation of growth hormone-binding protein secretase

“…3B), supporting the suggestion that the inhibition of PMA-induced GHBP shedding by these inhibitors may be a direct result of inhibition of proteolytic cleavage of GHR at the cell surface. Cell-permeable sulfhydryl-reactive alkylating reagents, such as iodoacetamide or NEM, were previously shown to promote proteolytic cleavage of GHBP (Trivedi & Daughaday 1988, Massa et al 1993, Amit et al 1994, Harrison et al 1995, Bick et al 1996, Alele et al 1998. Thus, it was important to test whether BB-3103 and Ro31-9790 could also inhibit NEM-induced GHBP shedding from the CHO/hGHR cell line, since another metalloprotease inhibitor, IC3, was shown to inhibit GHBP shedding from IM-9 cells (Alele et al 1998).…”

“…We and others have demonstrated that sulfhydryl-reactive agents markedly induced GHBP release from IM-9 human lymphocytes (Trivedi & Daughaday 1988, Massa et al 1993, Alele et al 1998, Hep G2 human hepatoma cells (Amit et al 1994, Harrison et al 1995 and Chinese hamster ovary (CHO) cells transfected with rabbit or human GHR (hGHR) (Bick et al 1996, Amit et al 1999. We then suggested that the increased release of GHBP might be a consequence of alkylation of one or more free sulfhydryl group(s) on an endopeptidase that apparently becomes activated to induce GHBP shedding (Amit et al 1999).…”

“…In the human and rabbit, growth hormone (GH) bindingprotein (GHBP) is generated by proteolytic cleavage of the full-length transmembrane GH receptor (GHR) (Leung et al 1987, Trivedi & Daughaday 1988, Sotiropoulos et al 1993. Furthermore, an alternatively spliced form of human (h) GHR was demonstrated to encode a cytoplasmically truncated isoform of hGHR (hGHR tr ) and to regulate GHBP generation (Dastot et al 1996, Amit et al 1997, Ross et al 1997.…”

Ontogeny of growth hormone receptor gene expression in tissue of growth-selected strains of broiler chickens

“…In the mouse, GHR and GHBP mRNA with alternative 5 -UTRs are differentially expressed in mouse liver and placenta (Southard et al 1995). In humans and rabbits, the circulating GHBP probably arises from the proteolytic cleavage of the membrane receptors (Leung et al 1987, Trivedi & Daughaday 1988, Sotiropoulos et al 1993, Bingham et al 1994. In the chicken, it appears that the serum GHBP is produced from proteolytic cleavage of the membrane receptors rather than from alternative splicing of the cGHR gene (Cogburn et al 1997).…”

The Clinical Significance of Growth Hormone Binding Protein

“…Based on a study with the IM9 lymphoma cell line, which exhibits somatogenic receptors for GH, it was postulated that the extracellular domain of the GH-receptor is shed from the cell by a limited proteolytic cleavage [7]. On the other hand, separate mRNAs were ascribed for GH-BP and the GH-receptor in the mouse [8] and rat [5], probably generated by alternative splicing of DNA.…”