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 facto r (ARF) nucleotide-binding site opener (ARNO) in desensitization of the lut einizing hormone/choriogonadotropin (LH/CG) receptor, independent of recept or internalization, using a cell-free plasma membrane model. We recently sh owed that the addition of recombinant ARNO promotes binding of beta -arrest in1 to the third intracellular (3i) loop of the active LH/CG receptor, ther eby reducing the ability of the receptor to activate the stimulatory G prot ein and signal to adenylyl cyclase. In the present report we determined whe ther ARNO is detectable in follicular membranes and whether the catalytical ly inactive E156K ARNO mutant, containing a mutation in the Sec7 domain, ca n act in a dominant negative manner to block LH/CG receptor desensitization . Results show that ARNO is readily detected in follicular membranes and th at levels of membrane-associated ARNO increase with follicular maturation. The addition of catalytically inactive E156K ARNO blocks both the release o f beta -arrestin1 from its membrane docking site, based on Western blot ana lysis, and development of LH/CG receptor desensitization. We also investiga ted whether a point mutation in the pleckstrin homology (PH) domain of ARNO (R280D), which blocks binding of phosphoinositides like phosphatidylinosit ol 3,4,5-trisphosphate and phosphatidylinositol 4,5-bisphosphate (PIP2) but not catalytic activity, disrupts LH/CG receptor desensitization. R280D ARN O neither promotes nor inhibits LH/CG receptor desensitization, consistent with a requirement of the PH domain of ARNO for its association with the pl asma membrane. LH/CG receptor activation of ARNO is not mediated by activat ion of phosphatidylinositol 3-kinase (PI 3-kinase) or by G protein beta gam ma subunits. Taken together, these results suggest that LH/CG receptor prom otes beta -arrestin1 release from its membrane docking site to bind to the 3i loop of the LH/CG receptor via activation of membrane delimited endogeno us ARNO. As ARNO activation is independent of PI 3-kinase and G beta gamma, our results are consistent with a role for PIP2 in receptor-stimulated ARN O activation.