The human growth hormone (GH) receptor and its truncated isoform: Sulfhydryl group inactivation in the study of receptor internalization and GH-binding protein generation

The human GH receptor (hGHR) contains nine intracellular and seven extracel lular cysteines, of which six are linked by disulfide bonds and one, at pos ition 241 proximal to the membrane, is free. Recently, an alternatively spl iced GHR isoform has been isolated; it encodes a truncated receptor lacking most of the cytoplasmic domain (hGHRtr). In the present study, we have exa mined the effect of sulfhydryl group(s) inactivation on receptor internaliz ation and GH binding-protein (GHBP) generation from the human (h) and rabbi t (rb) full-length GHR, as well as from hGHRtr and a mutant of the free ext racellular cysteine (hGHRtr-C241A), expressed in Chinese hamster ovary (CHO ) cells. in CHO/rbGHR and CHO/hGHR cells, permeable sulfhydryl-reactive age nts, like N-ethylmaleimide (NEM) and iodacetamide (LA), inhibited GHR inter nalization and induced an immediate dose-dependent loss of cellular GHR, as sociated with a concomitant marked increase in released GHBP. In contrast, the membrane impermeable IA derivative A-484 had no effect on either GHBP r elease or on GHR internalization. NEM exposure of CHO cells, expressing hGH Rtr, resulted in a dose-dependent increase in GHBP generation, but only a m oderate decrease in cellular hGHRtr. The importance of the only unpaired cy steine in these processes was evaluated in CHO/hGHRtr-C241A cells. hGHRtr-C 241A was similar to hGHRtr in its impaired internalization and Enhanced GHB P release by NEM. Taken together, these data suggest that intracellular sulmydryl groups, wit hin membranal endocytic vesicles, that do not belong to the GHR molecule, a re involved in receptor internalization and GHBP generation. In addition, t he present study demonstrates that despite impaired hGHR internalization/do wn-regulation, the inducible release of GHBP was not affected, further sugg esting that GBR endocytosis is not a prerequisite for GHBP generation.