Desensitization of the luteinizing hormone/choriogonadotropin receptor in ovarian follicular membranes is inhibited by catalytically inactive ARNO(+).
We have investigated the participation of endogenous ADP-ribosylation factor (ARF) nucleotide-binding site opener (ARNO) in desensitization of the luteinizing hormone/choriogonadotropin (LH/CG) receptor, independent of receptor internalization, using a cell-free plasma membrane model. We recently showed that the addition of recombinant ARNO promotes binding of beta-arrestin1 to the third intracellular (3i) loop of the active LH/CG receptor, thereby reducing the ability of the receptor to activate the stimulatory G protein and signal to adenylyl cyclase. In the present report we determined whether ARNO is detectable in follicular membranes and whether the catalytically inactive E156K ARNO mutant, containing a mutation in the Sec7 domain, can act in a dominant negative manner to block LH/CG receptor desensitization. Results show that ARNO is readily detected in follicular membranes and that levels of membrane-associated ARNO increase with follicular maturation. The addition of catalytically inactive E156K ARNO blocks both the release of beta-arrestin1 from its membrane docking site, based on Western blot analysis, and development of LH/CG receptor desensitization. We also investigated whether a point mutation in the pleckstrin homology (PH) domain of ARNO (R280D), which blocks binding of phosphoinositides like phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 4,5-bisphosphate (PIP(2)) but not catalytic activity, disrupts LH/CG receptor desensitization. R280D ARNO neither promotes nor inhibits LH/CG receptor desensitization, consistent with a requirement of the PH domain of ARNO for its association with the plasma membrane. LH/CG receptor activation of ARNO is not mediated by activation of phosphatidylinositol 3-kinase (PI 3-kinase) or by G protein beta gamma subunits. Taken together, these results suggest that LH/CG receptor promotes beta-arrestin1 release from its membrane docking site to bind to the 3i loop of the LH/CG receptor via activation of membrane delimited endogenous ARNO. As ARNO activation is independent of PI 3-kinase and G beta gamma, our results are consistent with a role for PIP(2) in receptor-stimulated ARNO activation.
Mukherjee
S
S
Department of Cell and Molecular Biology, Northwestern University Medical School, Chicago, Illinois 60611, USA.
Casanova
J E
JE
Hunzicker-Dunn
M
M
D000595:Amino Acid Sequence; D000818:Animals; D019390:Arrestins / Q000378:metabolism; D002384:Catalysis; D005260:Female; D020690:GTPase-Activating Proteins / Q000032:analysis / Q000737:chemistry / Q000502:physiology*; D006160:Guanosine Triphosphate / Q000378:metabolism; D008969:Molecular Sequence Data; D006080:Ovarian Follicle / Q000502:physiology*; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000096:biosynthesis; D011974:Receptors, LH / Q000037:antagonists & inhibitors / Q000502:physiology*; D013552:Swine; D000071557:beta-Arrestins
D019390:Arrestins; D020690:GTPase-Activating Proteins; D019269:Phosphatidylinositol 4,5-Diphosphate; D011974:Receptors, LH; D000071557:beta-Arrestins; C102474:cytohesin-2; D006160:Guanosine Triphosphate