Activation of the luteinizing hormone/choriogonadotropin hormone receptor promotes ADP ribosylation factor 6 activation in porcine ovarian follicular membranes.
Previously we demonstrated in a cell-free ovarian follicular plasma membrane model that agonist-dependent desensitization of the luteinizing hormone/choriogonadotropin receptor (LH/CG R) is GTP-dependent, mimicked by the addition of ADP-ribosylation factor (ARF) nucleotide binding site opener, which acts as a guanine nucleotide exchange factor for ARFs 1 and 6, and selectively inhibited by synthetic N-terminal ARF6 peptides. We therefore sought direct evidence that activation of the LH/CG R promotes activation of ARF1 and/or ARF6. Using a classic ARF activation assay, the cholera toxin-catalyzed ADP-ribosylation of G alpha(s), results show that LH/CG R activation stimulates an ARF protein by a brefeldin A-independent mechanism. Synthetic N-terminal inhibitory ARF6 but not ARF1 peptide blocks LH/CG R-stimulated ARF activity. LH/CG R activation also promotes the binding of a photoaffinity GTP analog to a protein that migrates on one- and two-dimensional polyacrylamide gel electrophoresis with ARF6. These results suggest that ARF6 is the predominant ARF activated by the LH/CG R. To activate ARF6, the LH/CG R does 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 terminus 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 levels by catalytically inactive ARF nucleotide binding-site opener. These results provide direct evidence that LH/CG R activation leads to the activation of membrane-delimited ARF6.
Salvador
L M
LM
Department of Cell and Molecular Biology, Northwestern University Medical School, Chicago, Illinois 60611, USA.
Mukherjee
S
S
Kahn
R A
RA
Lamm
M L
ML
Fazleabas
A T
AT
Maizels
E T
ET
Bader
M F
MF
Hamm
H
H
Rasenick
M M
MM
Casanova
J E
JE
Hunzicker-Dunn
M
M
D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000378:metabolism*; D000818:Animals; D020126:Brefeldin A / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism*; D002772:Cholera Toxin / Q000494:pharmacology; D006063:Chorionic Gonadotropin / Q000494:pharmacology; D003902:Detergents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005260:Female; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D017830:Octoxynol / Q000494:pharmacology; D006080:Ovarian Follicle / Q000378:metabolism; D010053:Ovary / Q000166:cytology*; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D011974:Receptors, LH / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013552:Swine
D000089982:ADP-Ribosylation Factor 6; D006063:Chorionic Gonadotropin; D003902:Detergents; D020662:Guanine Nucleotide Exchange Factors; D010455:Peptides; D011974:Receptors, LH; D011994:Recombinant Proteins; D020126:Brefeldin A; D006160:Guanosine Triphosphate; D017830:Octoxynol; D002772:Cholera Toxin; D006861:Hydrogen Peroxide; D020727:ADP-Ribosylation Factors; C000715348:ARF6 protein, human