Abstract
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.
Highlights
The binding of saturating concentrations of agonist to guanine nucleotide-binding (G) protein-coupled receptors initially results in productive coupling to an effector, resulting in increased effector activity
luteinizing hormone/ choriogonadotropin (LH/CG) receptor activation of ARNO is not mediated by activation of phosphatidylinositol 3-kinase (PI 3-kinase) or by G protein ␥ subunits. These results suggest that LH/CG receptor promotes -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
Based on our evidence that the addition of exogenous ARNO in the presence of 1 M GTP frees a pool of -arrestin1 to bind to the 3i loop of the activated LH/CG receptor to block receptor interaction with Gs [6], we hypothesized that LH/CG receptordependent activation of ADP-ribosylation factor 6 (ARF6) might be mediated by activation of endogenous, membrane-delimited ARNO
Summary
The binding of saturating concentrations of agonist to guanine nucleotide-binding (G) protein-coupled receptors initially results in productive coupling to an effector, resulting in increased effector activity. The addition of catalytically inactive E156K ARNO blocks both the release of -arrestin1 from its membrane docking site, based on Western blot analysis, and development of LH/CG receptor desensitization.
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