Control of growth hormone (GH) binding protein release from human hepatoma cells expressing full-length GH receptor.
In humans and rabbits, the circulating GH binding protein (GHBP) is released from the GH receptor by cleavage at a site proximal to the cell surface. There is evidence that GHBP status is predictive of GH responsiveness, presumably because it reflects GH receptor status. This assumes that GHBP release is not a regulated step. Here we report a model for study of GHBP release that provides some insight into this question. Human HepG2 cells were stably transfected with rabbit GH receptor and shown to be responsive to nonprimate (bovine) GH, indicating functionality of the transfected receptor. These cells released GHBP of the expected size, and this release could be increased by incubation with a phorbol ester, which stimulated receptor synthesis through the cytomegalovirus promoter. We surveyed a wide range of protease inhibitors both with and without streptolysin-O permeabilization, with the intention of defining the endogenous protease. Of 16 inhibitors, only benzamidine proved an effective inhibitor of release, indicating the existence of a novel protease. We could increase GHBP release with a membrane impermeable thiol blocker, suggesting activation of a membrane protease. We examined the ability of IGF-1, insulin, dexamethasone, sex steroids, and T4 to influence GHBP release. Although these agents are known to be effective in the parent hepatoma line, none were effective in modulating GHBP release, although GH itself decreased release by around 30% as assessed with a ligand immunofunctional assay. We conclude that GHBP release appears to be constitutive in this model and driven by receptor availability. This is consistent with an in vivo situation where circulating GHBP provides an index of hepatic receptor expression.
Department of Physiology and Pharmacology, University of Queensland, St. Lucia, Australia.
S M
SM
Harrison
R
R
Barnard
K Y
KY
Ho
I
I
Rajkovic
M J
MJ
Waters
D000818:Animals; D001550:Benzamidines / Q000494:pharmacology; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism*; D002417:Cattle; D013006:Growth Hormone / Q000378:metabolism*; D010703:Phorbol Esters / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology*; D011986:Receptors, Somatotropin / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured
D001550:Benzamidines; D002352:Carrier Proteins; D010703:Phorbol Esters; D011480:Protease Inhibitors; D011986:Receptors, Somatotropin; D011994:Recombinant Proteins; D013006:Growth Hormone; C032157:benzamidine; C051815:somatotropin-binding protein