Activation of the luteinizing hormone/choriogonadotropin hormone receptor promotes ADP ribosylation factor 6 activation in porcine ovarian follicularmembranes

Previously we demonstrated in a cell-free ovarian follicular plasma membran e model that agonist-dependent desensitization of the luteinizing hormone/c horiogonadotropin receptor (LH/CG R) is GTP-dependent, mimicked by the addi tion of ADP-ribosylation factor (ARF) nucleotide binding site opener, which acts as a guanine nucleotide exchange factor for ARFs 1 and 6, and selecti vely inhibited by synthetic N-terminal ARF6 peptides. We therefore sought d irect evidence that activation of the LH/CG R promotes activation of ARF1 a nd/or ARF6. Using a classic ARF activation assay, the cholera toxin-catalyz ed ADP-ribosylation of G alpha (s), results show that LH/CG R activation st imulates an ARF protein by a brefeldin A-independent mechanism. Synthetic N -terminal inhibitory ARF6 but not ARF1 peptide blocks LH/CG R-stimulated AR F activity. LH/CG R activation also promotes the binding of a photoaffinity GTP analog to a protein that migrates on one- and two-dimensional polyacry lamide gel electrophoresis with ARF6. These results suggest that ARF6 is th e predominant ARF activated by the LH/CG R. To activate ARF6, the LH/CG R d oes not appear to signal through the C-terminal regions of G alpha (i) or G alpha (q) or through the second or third intracellular loops or the N term inus of the cytoplasmic tail of the LH/CG R. Although exogenous recombinant ARNO promotes only a small increase in ARF6 activation in the presence of activated LH/CG R, hCG-stimulated ARF6 activation is reduced to basal level s by catalytically inactive ARF nucleotide binding-site opener. These resul ts provide direct evidence that LH/CG R activation leads to the activation of membrane-delimited ARF6.