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Luteinizing Hormone/Choriogonadotropin-Dependent, Cholera Toxin-Catalyzed Adenosine 5'-Diphosphate (ADP)-Ribosylation of the Long and Short Forms of G_s and Pertussis Toxin-Catalyzed ADP-Ribosylation of Gi

Department of Cell and Molecular Biology (R.M.R.-G., M.H.-D) Northwestern University Medical School Chicago, Illinois 60611
Department of Physiology and Biophysics and Psychiatry (M.M.R.) University of Illinois College of Medicine Chicago, Illinois 60680

Although it is well established that activated LH/human (h) CG receptor stimulates adenylyl cyclase activity (via the heterotrimeric stimulatory guanine nucleotide-binding protein, G_s) and in some cells stimulates phospholipase C activity, there is no evidence for a direct physical interaction between the LH/CG receptor and G_s or any other G protein(s). We conducted studies using cholera toxin (CTX) and pertussis toxin (PTX) to determine which G proteins were associated with the LH/CG receptor in ovarian follicular membranes. Since hormone-dependent, CTX-catalyzed ADP ribosylation (AR) constitutes evidence that a G protein is specifically associated with a receptor, CTX-catalyzed AR of membrane proteins was examined both in the presence and absence of guanine nucleotides to determine which G proteins exhibit hCG-dependent labeling by [³²P]NAD. Results demonstrated the time- and hCG-dependent AR of both a 45-kDa protein and a 48/50-kDa doublet as well as a 40-kDa protein that was also sensitive to AR by PTX in a time- and hCG-dependent manner. Using anti-G protein antisera to specifically immunoprecipitate photoaffinity-labeled G proteins, we were able to identify the 45- and 48/50 kDa proteins as the short and long forms of G_s and the 40-kDa protein as G_i. A monoclonal anti-hCG antibody immunoprecipitated the activated LH/CG receptor along with the long and short forms of G_s and G_i. These results suggest that a portion of G_i along with the long and short forms of G_s are associated physically with the LH/CG receptor in ovarian follicular membranes.

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