CHARACTERIZATION AND PURIFICATION OF LH/HCG RECEPTORS AND ADENYLATE CYCLASE

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1) The LH/hCG receptor from rat ovary and testis has been purified by sequential affinity column chromatography and isolated as a single protein species on SDS-PAGE under reducing conditions. The purified testicular receptor was shown to be phosphorylated in vitro by the catalytic subunit of cAMP-dependent protein kinase. Occupancy of the receptors by hCG significantly increased the rate of phosphorylation by the catalytic subunit of cAMP-dependent protein kinase, while maximal stoichiometry of phosphorylation was not affected by hCG. However, prolonged preincubation of hCG with the receptor reduced the rate of receptor phosphorylation. Identical phosphopeptide maps were obtained by reverse phase FPLC following trypsinization of both phosphorylated receptors. Six peaks contained phosphoserine and the major component was also phosphorylated on threonine. The phosphorylated receptor, like the native receptor, bound wheat germ lectin and hCG-Sepharose, and migrated as a single band of Mr=90,000 (testis) and Mr = 85,000 (ovary) respectively on SDS-PAGE. Neuraminidase treatment of purified receptors caused reductions in Mr to 82,000 plus/equal to 3,400 (testis) and 77,000 plus/equal to 3,700 (ovary), and further treatment with O-glycanase had little effects on molecular size. However, deglycosylation with N-glycosidase and Endoglycosidase F produced a single labeled polypeptide of Mr=59,000 plus/equal to 3,000 for both receptors. These results indicate that LH/hCG receptors are sialoglycoproteins with predominately N-linked glycosylation, and suggest that changes in the glycosylation pattern could account for the size difference between testicular and ovarian receptors. The various enzyme treatment also suggested that the LH/hCG receptor contains sialylated N-linked carbohydrate chains of the biantennary and/or hybrid type. Our studies indicate that receptor occupancy by hCG leads to a conformational change which facilitates its phosphorylation during initial binding and reduces the rate of phosphorylation after more prolonged exposure to gonadotropin. 2) The Nb2 lymphoma cell line which is dependent upon lactogen for proliferation was used to initiate studies on the transduction mechanism of prolactins action. cAMP modified PRL-stimulated Nb2 lymphoma cell mitogenesis. The differences between the effects of pertussis toxin and cholera toxin (i.e. biphasic effect, degree of inhibition) and also the differential effect of PMA on Nb2 cell replication suggests the involvement of one or more G protein in PRL action or its modulation, including a cAMP-independent mechanism.