Involvement of plasma membrane enzymes in the proteolytic cleavage of luteinizing hormone receptor.

In vitro degradation of LH receptor after occupancy by hCG was studied. Rat ovarian membranes labeled with [125I]iodo-hCG were incubated at 37 C; as a result, 30-40% of the radioactivity initially bound was rendered soluble in the medium. The molecular complexes in the medium and in incubated membranes solubilized with 1% Triton X-100 were then cross-linked with glutaraldehyde and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Particulate receptor-[125I]iodo-hCG complex exhibiting an apparent mol wt of 125,000 was cleaved during incubation into two distinct components (mol wt, 96,000 and 74,000) which appeared in the medium. Using tritiated hCG (beta-subunit labeled) instead of radioiodinated hCG (alpha-subunit labeled), these same two components were also observed, indicating that they both contain intact hCG (alpha and beta) as a part of their structure. In addition to the hormone (mol wt, 48,000), these two components contain receptor fragments with mol wt of 64,000 or 38,000, demonstrated directly by labeling the particulate receptor itself with periodate-tritiated borohydride before tagging with unlabeled hCG and in vitro incubation. These receptor fragments were purified from the medium by hCG-directed immunoaffinity chromatography and detached from the hormone by pH treatment. The intact receptor extracted from the membranes with detergent and purified identically in the absence of proteolysis migrated as a 90,000 mol wt polypeptide. These results demonstrate that after hormone occupancy, proteolytic cleavage of the 90,000 mol wt receptor polypeptide occurs at two specific sites. Thiol-blocking agents selectively prevented the appearance of the larger component (hCG coupled to 64,000 mol wt receptor fragment), while metal-chelating agents markedly decreased the appearance of the smaller component (hCG coupled to 38,000 mol wt receptor fragment) in the medium. Identical observations, obtained upon incubation of plasma membranes purified by sucrose density gradient centrifugation, suggest that plasma membrane enzymes are involved.