Photoaffinity labeling of the gonadotropin receptor with native, asialo, and deglycosylated choriogonadotropin.
Human choriogonadotropin (hCG) is a heterodimeric hormone composed of an alpha and a beta subunit. hCG and its asialo (ashCG) and deglycosylated (dghCG) forms vary in their ability to stimulate hormone responsive adenylate cyclase. ashCG is a partial agonist, and dghCG is an antagonist. Photoactivatable moieties were coupled to hCG, ashCG, and dghCG, and the derivatives were radioiodinated. Competitive binding studies indicate that all of the derivatives had a similar affinity for the gonadotropin receptor on porcine granulosa cell membranes. Radiolabeled derivatives were used to photoaffinity label the gonadotropin receptor. Radiolabeled complexes were separated by NaDodSO4/PAGE. All of the derivatives produced similar autoradiographic patterns, except that dghCG produced an additional 48-kDa complex. To investigate the structure of the complexes further, peptide mapping of proteolytic digests was used. All, except for the 48-kDa complex, generated similar peptide maps indicating a relationship between those complexes in which the smaller components are part of the larger. The 48-kDa complex contained both subunits of 40-kDa dghCG. Therefore, this complex is expected to contain an additional component of 8 kDa. The complex was generated whether the hormone-receptor complex was photoaffinity labeled on cells, on isolated membranes, or after solubilizing in detergent. Formation was blocked by excess hCG and did not occur in the absence of UV irradiation. We conclude that the hCG derivatives are able to photoaffinity label the hCG receptor but that the dghCG derivative can photoaffinity label an additional component that was not observed when derivatives of hCG or ashCG were used to label the receptor.
D000262:Adenylyl Cyclases / Q000378:metabolism; D000345:Affinity Labels; D000818:Animals; D001212:Asialoglycoproteins / Q000378:metabolism*; D006063:Chorionic Gonadotropin / Q000378:metabolism*; D005260:Female; D006107:Granulosa Cells / Q000378:metabolism; D007700:Kinetics; D010446:Peptide Fragments / Q000032:analysis; D011956:Receptors, Cell Surface / Q000378:metabolism*; D011966:Receptors, LHRH; D013329:Structure-Activity Relationship; D013552:Swine
D000345:Affinity Labels; D001212:Asialoglycoproteins; D006063:Chorionic Gonadotropin; D010446:Peptide Fragments; D011956:Receptors, Cell Surface; D011966:Receptors, LHRH; D000262:Adenylyl Cyclases