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Desensitization of the Luteinizing Hormone/Choriogonadotropin Receptor in Ovarian Follicular Membranes Is Inhibited by Catalytically Inactive ARNO+

2001; Elsevier BV; Volume: 276; Issue: 9 Linguagem: Inglês

10.1074/jbc.c000725200

ISSN

1083-351X

Autores

Sutapa Mukherjee, James E. Casanova, Mary Hunzicker-Dunn,

Tópico(s)

Adenosine and Purinergic Signaling

Resumo

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 β-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 β-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 (PIP2) 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 βγ subunits. Taken together, these results suggest that LH/CG receptor promotes β-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 Gβγ, our results are consistent with a role for PIP2 in receptor-stimulated ARNO activation. 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 β-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 β-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 (PIP2) 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 βγ subunits. Taken together, these results suggest that LH/CG receptor promotes β-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 Gβγ, our results are consistent with a role for PIP2 in receptor-stimulated ARNO activation. luteinizing hormone/choriogonadotropin human choriogonadotropin ADP ribosylation factor ARF nucleotide binding site opener bovine serum albumin third intracellular loop stimulatory guanine nucleotide-binding protein guanine nucleotide exchange factor adenylyl cyclase phosphatidylinositol phosphatidylinositol 3,4,5-trisphosphate phosphatidylinositol 4,5-bisphosphate pleckstrin homology 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. Thereafter, effector activity often declines or becomes desensitized as a result of the uncoupling of the agonist-bound receptor from its cognate G protein (1Hausdorff W.P. Caron M.G. Lefkowitz R.J. FASEB. J. 1990; 4: 2881-2889Crossref PubMed Scopus (1087) Google Scholar). We have used a cell-free plasma membrane model to investigate the cellular mechanism of homologous luteinizing hormone/choriogonadotropin (LH/CG)1 receptor desensitization independent of LH/CG receptor internalization (2Bockaert J. Hunzicker-Dunn M. Birnbaumer L. J. Biol. Chem. 1976; 251: 2653-2663Abstract Full Text PDF PubMed Google Scholar,3Ekstrom R.C. Hunzicker-Dunn M. Endocrinology. 1989; 124: 956-963Crossref PubMed Scopus (27) Google Scholar). We have recently shown that desensitization of the LH/CG receptor requires the binding of membrane-delimited β-arrestin1 (Arrestin 2) to the third intracellular loop (3i) of the LH/CG receptor, resulting in reduced cAMP production (4Mukherjee S. Palczewski K. Gurevich V.V. Hunzicker-Dunn M. J. Biol. Chem. 1999; 274: 12984-12989Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 5Mukherjee S. Palczewski K. Benovic J.L. Gurevich V.V. Hunzicker-Dunn M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 493-498Crossref PubMed Scopus (42) Google Scholar). This conclusion is based on evidence that preincubation of membranes with neutralizing anti-arrestin antibodies prevents development of LH/CG receptor desensitization (5Mukherjee S. Palczewski K. Benovic J.L. Gurevich V.V. Hunzicker-Dunn M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 493-498Crossref PubMed Scopus (42) Google Scholar), that β-arrestin1 binds directly and selectively to a synthetic peptide corresponding to the 3i loop of the LH/CG receptor (4Mukherjee S. Palczewski K. Gurevich V.V. Hunzicker-Dunn M. J. Biol. Chem. 1999; 274: 12984-12989Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar), and that preincubation of membranes with a synthetic peptide corresponding to the 3i loop of the LH/CG receptor completely blocks development of receptor desensitization (4Mukherjee S. Palczewski K. Gurevich V.V. Hunzicker-Dunn M. J. Biol. Chem. 1999; 274: 12984-12989Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). We have also recently shown that LH/CG receptor activation not only exposes a binding site for β-arrestin1 at the 3i loop on the receptor but also leads to the apparent activation of the small G protein ADP-ribosylation factor 6 (ARF6), resulting in the release of a pool of β-arrestin1 from its membrane docking site (6Mukherjee S. Gurevich V.V. Jones J.C.R. Casanova J.E. Frank S.R. Maizels E.T. Bader M.-F. Kahn R.A. Palczewski K. Aktories K. Hunzicker-Dunn M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5901-5906Crossref PubMed Scopus (72) Google Scholar). These conclusions are based on the following results. (a) ARNO (25 nm), a guanine nucleotide exchange factor (GEF) for ARFs 1 and 6 (7Frank S. Upender S. Hansen S.H. Cassanova J.E. J. Biol. Chem. 1998; 273: 23-27Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 8Chardin P. Paris S. Antonny B. Robineau S. Beraud-Dufour S. Jackson C.L. Chabre M. Nature. 1996; 384: 481-484Crossref PubMed Scopus (409) Google Scholar), promotes desensitization of the LH/CG receptor in the presence but not the absence of GTP concomitant with the release of β-arrestin1 from its membrane docking site (6Mukherjee S. Gurevich V.V. Jones J.C.R. Casanova J.E. Frank S.R. Maizels E.T. Bader M.-F. Kahn R.A. Palczewski K. Aktories K. Hunzicker-Dunn M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5901-5906Crossref PubMed Scopus (72) Google Scholar). (b) Catalytically dead E156K ARNO mutant (at 25 or 50 nm) does not cause LH/CG receptor desensitization (6Mukherjee S. Gurevich V.V. Jones J.C.R. Casanova J.E. Frank S.R. Maizels E.T. Bader M.-F. Kahn R.A. Palczewski K. Aktories K. Hunzicker-Dunn M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5901-5906Crossref PubMed Scopus (72) Google Scholar). (c) Preincubation of membranes with synthetic N-terminal ARF6 peptide, but not with the corresponding ARF1 peptide, prevents the release of β-arrestin1 from its membrane docking site and thereby prevents development of LH/CG receptor desensitization (6Mukherjee S. Gurevich V.V. Jones J.C.R. Casanova J.E. Frank S.R. Maizels E.T. Bader M.-F. Kahn R.A. Palczewski K. Aktories K. Hunzicker-Dunn M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5901-5906Crossref PubMed Scopus (72) Google Scholar). LH/CG receptor-dependent activation of the small G protein ARF6 is consistent with earlier studies showing that LH/CG receptor desensitization exhibits an obligatory requirement for GTP (3Ekstrom R.C. Hunzicker-Dunn M. Endocrinology. 1989; 124: 956-963Crossref PubMed Scopus (27) Google Scholar,9Ekstrom R.C. Hunzicker-Dunn M. Endocrinology. 1989; 125: 2470-2474Crossref PubMed Scopus (27) Google Scholar, 10Ezra E. Salomon Y. J. Biol. Chem. 1980; 255: 653-658Abstract Full Text PDF PubMed Google Scholar, 11Ezra E. Salomon Y. J. Biol. Chem. 1981; 256: 5377-5382Abstract Full Text PDF PubMed Google Scholar). Based on our evidence that the addition of exogenous ARNO in the presence of 1 μm GTP frees a pool of β-arrestin1 to bind to the 3i loop of the activated LH/CG receptor to block receptor interaction with Gs (6Mukherjee S. Gurevich V.V. Jones J.C.R. Casanova J.E. Frank S.R. Maizels E.T. Bader M.-F. Kahn R.A. Palczewski K. Aktories K. Hunzicker-Dunn M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5901-5906Crossref PubMed Scopus (72) Google Scholar), we hypothesized that LH/CG receptor-dependent activation of ARF6 might be mediated by activation of endogenous, membrane-delimited ARNO. In the present report we therefore sought to determine whether endogenousmembrane-delimited ARNO participates in LH/CG receptor desensitization. All chemicals were from previously described sources (4Mukherjee S. Palczewski K. Gurevich V.V. Hunzicker-Dunn M. J. Biol. Chem. 1999; 274: 12984-12989Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 5Mukherjee S. Palczewski K. Benovic J.L. Gurevich V.V. Hunzicker-Dunn M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 493-498Crossref PubMed Scopus (42) Google Scholar). Recombinant ARNO and E156K ARNO were expressed and purified as described previously (7Frank S. Upender S. Hansen S.H. Cassanova J.E. J. Biol. Chem. 1998; 273: 23-27Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). A sucrose gradient-purified membrane fraction enriched in adenylyl cyclase (AC) activity was isolated from preovulatory-size porcine ovarian follicles and stored at −70 °C (6Mukherjee S. Gurevich V.V. Jones J.C.R. Casanova J.E. Frank S.R. Maizels E.T. Bader M.-F. Kahn R.A. Palczewski K. Aktories K. Hunzicker-Dunn M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5901-5906Crossref PubMed Scopus (72) Google Scholar). The two-stage desensitization reaction (4Mukherjee S. Palczewski K. Gurevich V.V. Hunzicker-Dunn M. J. Biol. Chem. 1999; 274: 12984-12989Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar) is summarized in the Fig. 2 legend. Supernatant and pellet fractions of porcine ovarian follicles were obtained by homogenizing tissue in 10 mm TRIS-HCl, pH 7.0, 1.0 mm EDTA with a glass-glass Dounce homogenizer (∼10 strokes) followed by centrifugation at 1,000 × g for 5 min and then at 10,000 × g for 30 min. The final pellet was resuspended in 1 volume of the original homogenate, and an aliquot of the final supernatant and pellet fractions was obtained for protein determination. SDS-STOP was then added, and samples were boiled for 10 min and stored at −70 °C. Western blotting with anti-β-arrestin antibody (Transduction Laboratories, Lexington, KY), the pan-arrestin antibody F4C1, and affinity-purified anti-ARNO antibody (7Frank S. Upender S. Hansen S.H. Cassanova J.E. J. Biol. Chem. 1998; 273: 23-27Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar) was performed as described previously (6Mukherjee S. Gurevich V.V. Jones J.C.R. Casanova J.E. Frank S.R. Maizels E.T. Bader M.-F. Kahn R.A. Palczewski K. Aktories K. Hunzicker-Dunn M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5901-5906Crossref PubMed Scopus (72) Google Scholar, 7Frank S. Upender S. Hansen S.H. Cassanova J.E. J. Biol. Chem. 1998; 273: 23-27Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). The results were analyzed using Student's t test (p < 0.05) (12Bender F.E. Douglass L.W. Kramer A. Statistical Methods for Food and Agriculture. AVI Publishing Co., Inc., Westport, CT1982Google Scholar). We first determined whether ARNO is detectable in follicular membranes enriched in AC activity. Results (Fig.1 A, lane 4) show that a band reactive with affinity purified anti-ARNO antibody (7Frank S. Upender S. Hansen S.H. Cassanova J.E. J. Biol. Chem. 1998; 273: 23-27Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar) is readily detectable in porcine ovarian follicular membranes and migrates on SDS-polyacrylamide gel electrophoresis somewhat faster than His6-tagged recombinant human ARNO (lanes 5–8). Quantitation of the amount of ARNO in partially purified ovarian follicular membranes by Western blotting, using the signal generated by recombinant ARNO as the standard, yielded a concentration of ∼1.5 μg of ARNO protein (or 32 nmol)/mg of membrane protein. The basis for the ARNO doublet (see Fig. 1 A, lanes 1–3) is not known but might correspond to phosphorylated and unphosphorylated ARNO (13Santy L.C. Frank S.R. Hatfield J.C. Casanova J.E. Curr. Biol. 1999; 9: 1173-1176Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 14Frank S.R. Hatfield J.C. Casanova J.E. Mol. Biol. Cell. 1998; 9: 3133-3146Crossref PubMed Scopus (112) Google Scholar). We additionally determined whether the amount of ARNO present in unpurified 10,000 × g membrane and supernatant fractions is regulated by follicular maturation. Results (Fig.1 B) show that whereas ARNO levels are relatively low and unregulated in the supernatant fractions of small (1–2 mm) compared with large (8–10 mm) follicles, the levels of ARNO in the membrane fraction are higher (∼6-fold) in large preovulatory follicles enriched in LH/CG receptors compared with levels in small immature follicles that do not contain LH/CG receptors (15Channing C.P. Kammerman S. Biol. Reprod. 1974; 10: 179-198Crossref PubMed Scopus (77) Google Scholar). These results show that the expression of ARNO is increased with development of porcine follicles from an immature to a preovulatory phenotype and that the majority of ARNO is localized to the pellet fraction. Based on evidence that ARNO is readily detected in membranes of preovulatory-size follicles, we next sought to determine whether catalytically inactive ARNO could compete with endogenous ARNO to block agonist-dependent LH/CG receptor desensitization. The E156K mutation in the Sec7 domain of ARNO blocks the ability of ARNO to promote GTP exchange at ARF6 but does not disrupt its ability to bind phosphoinositides via its pleckstrin homology (PH) domain (14Frank S.R. Hatfield J.C. Casanova J.E. Mol. Biol. Cell. 1998; 9: 3133-3146Crossref PubMed Scopus (112) Google Scholar). Although preincubation of membranes with 50 (not shown) or 100 nm (Fig. 2 A) E156K ARNO did not affect the extent of LH/CG receptor desensitization (hCG/hCG conditions), preincubation of membranes with 200 nm E156K ARNO significantly reduced (p < 0.05) the extent of LH/CG receptor desensitization. Thus, when membranes were preincubated with 200 (Fig. 2 A), 300, or 400 (not shown) nm E156K ARNO, hCG-stimulated AC activity measured with hCG in stage 1 (i.e. desensitization conditions) was no longer significantly different from values measured with BSA in stage 1. The capability of E156K ARNO to block LH/CG receptor desensitization was lost when E156K ARNO was boiled (Fig.2 B). The addition of E156K ARNO directly to the 5-min AC assay did not affect basal or hCG-, forskolin-, or aluminum fluoride-stimulated AC activities (Fig. 2 C). This result shows that E156K ARNO does not have a nonspecific effect on AC activity or on the ability of the LH/CG receptor or Gs to activate AC. To ascertain whether E156K ARNO indeed blocks development of LH/CG receptor desensitization by preventing ARF6 activation, we determined whether E156K ARNO blocks the release of β-arrestin1 from its membrane docking site. In the absence of LH/CG receptor activation, β-arrestin1 was readily detectable in follicular membranes (Fig.2 D, lane 4), presumably bound at a docking site distinct from the LH/CG receptor (6Mukherjee S. Gurevich V.V. Jones J.C.R. Casanova J.E. Frank S.R. Maizels E.T. Bader M.-F. Kahn R.A. Palczewski K. Aktories K. Hunzicker-Dunn M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5901-5906Crossref PubMed Scopus (72) Google Scholar). Incubation of membranes with hCG resulted in the retention of a fraction of β-arrestin1 at the membrane (Fig. 2 D, lane 3). This fraction of β-arrestin1 was competed away with a synthetic peptide corresponding to the 3i loop of the LH/CG receptor (lane 2), consistent with the premise that LH/CG receptor activation results in the binding of a fraction of β-arrestin1 to the 3i loop of the activated LH/CG receptor (4Mukherjee S. Palczewski K. Gurevich V.V. Hunzicker-Dunn M. J. Biol. Chem. 1999; 274: 12984-12989Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 6Mukherjee S. Gurevich V.V. Jones J.C.R. Casanova J.E. Frank S.R. Maizels E.T. Bader M.-F. Kahn R.A. Palczewski K. Aktories K. Hunzicker-Dunn M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5901-5906Crossref PubMed Scopus (72) Google Scholar). However, when E156K ARNO was included in the membrane incubation mix with hCG and 3i peptide, this fraction of β-arrestin1 was retained in the membrane (lane 1), presumably at its membrane docking site as it was no longer competed away by the 3i peptide. These results suggested that exogenous catalytically dead E156K ARNO effectively competes with endogenous ARNO to block the ability of the activated LH/CG receptor to promote β-arrestin1 release and consequent LH/CG receptor desensitization. Based on these results we can therefore conclude that endogenous ARNO is activated to promote GTP exchange at ARF6 in response to LH/CG receptor activation. ARNO contains a C-terminal PH domain that can bind phosphatidylinositol 3,4,5-trisphosphate (PIP3), the product of PI(4,5)bisphosphatate 3-kinase (PI 3-kinase), with high selectivity and affinity (Kd∼85 nm (16Venkateswarlu K. Oatey P.B. Tavare J.M. Cullen P.J. Curr. Biol. 1998; 8: 463-466Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar)). A point mutation at R280D in the PH domain of ARNO results in a mutant protein that retains its catalytic activity to promote GTP exchange on ARF6 but cannot bind phosphoinositides (14Frank S.R. Hatfield J.C. Casanova J.E. Mol. Biol. Cell. 1998; 9: 3133-3146Crossref PubMed Scopus (112) Google Scholar). We have shown that, unlike catalytically active wild-type ARNO (at 25 nm), which promotes LH/CG receptor desensitization, R280D ARNO at 50 nm does not promote LH/CG receptor desensitization (6Mukherjee S. Gurevich V.V. Jones J.C.R. Casanova J.E. Frank S.R. Maizels E.T. Bader M.-F. Kahn R.A. Palczewski K. Aktories K. Hunzicker-Dunn M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5901-5906Crossref PubMed Scopus (72) Google Scholar). This result suggests that the binding of phosphoinositides to the PH domain of ARNO is obligatory for LH/CG receptor to promote activation of ARNO. We therefore sought to determine whether R280D ARNO could function like E156K ARNO in a dominant negative manner to block development of LH/CG receptor desensitization in response to receptor activation. Preincubation of follicular membranes with 200 (Fig.3 A) or 300 nm (not shown) R280D ARNO does not affect the ability of receptor activation to promote LH/CG receptor desensitization. Consistent with this result, preincubation of membranes with the PI 3-kinase inhibitor wortmannin at 100 nm does not reduce the extent of LH/CG receptor desensitization (Fig. 3 B). As some PH domains also bind the βγ subunits of activated G proteins (17Bottomley M.J. Salim K. Panayotou G. Biochim. Biophys. Acta. 1998; 1436: 165-183Crossref PubMed Scopus (110) Google Scholar), we tested whether a βγ inhibitor peptide (18Chen J. DeVivo M. Dingus J. Harry A. Li J. Sui J. Carty D.J. Blank J.L. Exton J.H. Stoffel R.H. Inglese J. Lefkowitz R.J. Logothetis D.E. Hildebrandt J.D. Iyengar R. Science. 1975; 268: 1166-1169Crossref Scopus (236) Google Scholar), corresponding to residues 956–982 of AC2, could block development of LH/CG receptor desensitization. The results (Fig. 3 C) show that preincubation of follicular membranes with 10 μm βγ-inhibitor peptide QEHA (QEHAQEPEROYMCHIGTMVEFAYALVGK), which blocks βγ-dependent stimulation of AC2, β-adrenergic receptor kinase, and phospholipase C (18Chen J. DeVivo M. Dingus J. Harry A. Li J. Sui J. Carty D.J. Blank J.L. Exton J.H. Stoffel R.H. Inglese J. Lefkowitz R.J. Logothetis D.E. Hildebrandt J.D. Iyengar R. Science. 1975; 268: 1166-1169Crossref Scopus (236) Google Scholar), does not affect LH/CG receptor desensitization. Higher concentrations of the QEHA peptide inhibited basal and hCG- and forskolin-stimulated AC activities (Fig.3 C and not shown). Taken together, these results indicate that PIP3 or βγ signaling through the PH domain of ARNO does not appear to be obligatory for the activated LH/CG receptor to promote GTP exchange at ARF. ARNO is detected in a purified membrane fraction enriched in AC activity obtained from preovulatory-size porcine ovarian follicles at a concentration of ∼32 nmol/mg of membrane protein. Moreover, the expression of ARNO appears to be regulated with follicular maturation. Higher levels of ARNO are detected in membranes of mature preovulatory (8–10 mm) porcine follicles, which express LH/CG receptors, compared with small (1–2 mm) follicles, which do not express LH/CG receptors (15Channing C.P. Kammerman S. Biol. Reprod. 1974; 10: 179-198Crossref PubMed Scopus (77) Google Scholar). That increased expression of ARNO correlates with the induction of LH/CG receptors is very interesting and suggests that the same stimulus that induces LH/CG receptors, namely follicle-stimulating hormone (19Hsueh A.J.W. Adashi E.Y. Jones P.B.C. Welsh Jr., T.H. Endocr. Rev. 1984; 5: 76-110Crossref PubMed Scopus (875) Google Scholar), might also increase expression of ARNO. These results further suggest that ARNO, in an apparently inactive conformation, is constitutively associated with the plasma membrane of preovulatory ovarian follicles. The conclusion that ARNO is present in an inactive conformation and is activated upon engagement of the LH/CG receptor is based not only on results presented in Fig. 2, showing that LH/CG receptor desensitization is blocked by E156K ARNO, but also on evidence that ARF activation, measured as cholera toxin-catalyzed ADP-ribosylation of the long form of Gαs, is negligible in the absence of LH/CG receptor activation (20Rajagopalan-Gupta R.M. Rasenick M.M. Hunzicker-Dunn M. Mol. Endocrinol. 1997; 11: 538-549PubMed Google Scholar). 2L. M. Salvador, S. Mukherjee, R. A. Kahn, M. L. Lamm, M.-F. Bader, H. Hamm, M. M. Rasenick, J. E. Casanova, and M. Hunzicker-Dunn, manuscript in preparation. The basis for the apparently constitutive membrane association of ARNO is not clear. However, in other cellular models there is also evidence that a large portion of total cellular ARNO can be associated constitutively with the plasma membrane in the absence of directed membrane-receptor activation both on overexpression in various cell lines (7Frank S. Upender S. Hansen S.H. Cassanova J.E. J. Biol. Chem. 1998; 273: 23-27Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar) and in chromaffin cells isolated from bovine adrenal glands (21Caumont A.-S. Vitale N. Gensse M. Galas M.-C. Casanova J.E. Bader M.-F. J. Biol. Chem. 2000; 275: 15637-15644Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). We have shown with the porcine follicular membrane model that catalytically inactive E156K ARNO acts in a dominant negative manner to selectively block the development of LH/CG receptor desensitization in response to LH/CG receptor activation. Catalytically inactive E156K ARNO blocks the obligatory release of β-arrestin1 from its membrane docking site. As a result, the LH/CG receptor remains active to signal to Gs and AC despite the continued presence of saturating concentrations of receptor agonist. This result suggests that LH/CG receptor desensitization requires the activation of endogenous ARNO. However, based on the recent identification of the exchange factor for ARF6 (EFA6) (22Franco M. Peters P.J. Boretto J. van Donselaar E. neri A. D'Souza-Schorey C. Chavrier P. EMBO J. 1999; 18: 1480-1491Crossref PubMed Scopus (236) Google Scholar), in which the guanine nucleotide exchange activity is also insensitive to brefeldin A, we cannot rule out the possibility that the apparent dominant negative effect of E156K ARNO is attributable to its potential ability to sequester available ARF6, thus indirectly blocking LH/CG receptor desensitization potentially mediated by EFA6. ARNO, along with GRP1 and cytohesin-1, comprise a subfamily of guanine nucleotide exchange factors for the ARFs in which the guanine nucleotide exchange activity is not inhibited by the fungal metabolite brefeldin A (7Frank S. Upender S. Hansen S.H. Cassanova J.E. J. Biol. Chem. 1998; 273: 23-27Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 8Chardin P. Paris S. Antonny B. Robineau S. Beraud-Dufour S. Jackson C.L. Chabre M. Nature. 1996; 384: 481-484Crossref PubMed Scopus (409) Google Scholar, 23Nagel W. Zeitlmann L. Schilcher P. Geiger C. Kolanus J. Kolanus W. J. 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Consistent with these results, transfection of cells with ARNO or cytohesin-1 containing a mutation in the PH domain disrupts the plasma membrane association of these proteins and consequent biological responses, namely cytohesin-1-dependent β2 integrin adhesion to adhesion molecule 1 in Jurkat E6 leukemia cells (23Nagel W. Zeitlmann L. Schilcher P. Geiger C. Kolanus J. Kolanus W. J. Biol. Chem. 1998; 273: 14853-14861Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar) and ARNO-dependent actin reorganization in HeLa cells (14Frank S.R. Hatfield J.C. Casanova J.E. Mol. Biol. Cell. 1998; 9: 3133-3146Crossref PubMed Scopus (112) Google Scholar). The predominant ligands that have been shown to bind to the PH domains of these GEFs are phosphatidylinositol 4,5-bisphosphate (PIP2) and PIP3 (31Jackson C.L. Casanova J.E. Trends Cell Biol. 2000; 10: 60-67Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar). Although there have been seemingly contradictory results on the affinities of the PH domains of these proteins for PIP2 or PIP3, recent evidence from the Czech laboratory (32Klarlund J. Tsiaras W. Holik J.J. Chawla A. Czech M.P. J. Biol. Chem. 2000; 275: 32816-32821Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar) has resolved this apparent controversy by showing that ARNO, GRP1, and cytohesin-1 can exhibit either high selectivity for PIP3 or equivalent selectivity for PIP3 and PIP2 depending on whether the PH domain contains a diglycine or triglycine motif, respectively. Moreover, isoforms of each of the proteins in this subfamily appear to exist that express either the diglycine or triglycine motif even in the same tissue (7Frank S. Upender S. Hansen S.H. Cassanova J.E. J. Biol. Chem. 1998; 273: 23-27Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 8Chardin P. Paris S. Antonny B. Robineau S. Beraud-Dufour S. Jackson C.L. Chabre M. Nature. 1996; 384: 481-484Crossref PubMed Scopus (409) Google Scholar, 33Ogasawara M. Kim S.-C. Adamik R. Togawa A. Ferrans V.J. Takeda K. Kirby M. Moss J. Vaughan M. J. Biol. Chem. 2000; 275: 3221-3230Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 34Kim H.S. Chen Y. Lonai P. FEBS Lett. 1998; 433: 312-316Crossref PubMed Scopus (45) Google Scholar). Consistent with this result, recruitment and activation of these GEFs can depend on either PIP2 or PIP3, contingent upon whether the diglycine or triglycine motif in the PH domain is present (8Chardin P. Paris S. Antonny B. Robineau S. Beraud-Dufour S. Jackson C.L. Chabre M. Nature. 1996; 384: 481-484Crossref PubMed Scopus (409) Google Scholar, 13Santy L.C. Frank S.R. Hatfield J.C. Casanova J.E. Curr. Biol. 1999; 9: 1173-1176Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 24Klarlund J.K. Guilherme A. Holik J.J. Virbasius J.V. Chawla A. Czech M.P. Science. 1997; 275: 1927-1930Crossref PubMed Scopus (371) Google Scholar,28Paris S. Beraud-Dufour S. Robineau S. gay J. Antonny B. Chabre M. Chardin P. J. Biol. Chem. 1997; 272: 22221-22226Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 35Kharlund J.K. Rameh L.E. Cantley L.C. Buxton J.M. Holik H.H. Sakelis C. Patki V. Corvera S. Czeck M.P. J. Biol. Chem. 1998; 273: 1859-1862Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). In those cases in which GEF recruitment and activation rely on PIP3, these responses can be readily blocked by PI 3-kinase inhibitors like wortmannin (16Venkateswarlu K. Oatey P.B. Tavare J.M. Cullen P.J. Curr. Biol. 1998; 8: 463-466Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar, 23Nagel W. Zeitlmann L. Schilcher P. Geiger C. Kolanus J. Kolanus W. J. Biol. Chem. 1998; 273: 14853-14861Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 24Klarlund J.K. Guilherme A. Holik J.J. Virbasius J.V. Chawla A. Czech M.P. Science. 1997; 275: 1927-1930Crossref PubMed Scopus (371) Google Scholar, 30Langille S.E. Patki V. Klarlund J.K. Buxton J.M. Holik J.J. Chawla A. Corvera S. Czech M.P. J. Biol. Chem. 1999; 274: 27099-27104Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). There is also evidence for ARNO and cytohesin-1 that the adjacent C-terminal polybasic c domain cooperates with the PH domain to bind these GEFs tightly to the membrane, possibly by binding a phospholipid with a negative charge (8Chardin P. Paris S. Antonny B. Robineau S. Beraud-Dufour S. Jackson C.L. Chabre M. Nature. 1996; 384: 481-484Crossref PubMed Scopus (409) Google Scholar,13Santy L.C. Frank S.R. Hatfield J.C. Casanova J.E. Curr. Biol. 1999; 9: 1173-1176Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). LH/CG receptor desensitization is not inhibited by a point mutation in the PH domain of ARNO, which blocks its binding to all phosphoinositides. One interpretation of these results is that LH/CG receptor-dependent activation of ARNO is independent of the PH domain of ARNO. If phosphoinositide binding to the PH domain is not necessary for ARNO activation, then one would predict that R280D ARNO, like wild-type recombinant ARNO, should promote LH/CG receptor desensitization, since R280D ARNO retains its GTP exchange activity (14Frank S.R. Hatfield J.C. Casanova J.E. Mol. Biol. Cell. 1998; 9: 3133-3146Crossref PubMed Scopus (112) Google Scholar). Yet, R280D ARNO does not promote LH/CG receptor desensitization (Ref. 6Mukherjee S. Gurevich V.V. Jones J.C.R. Casanova J.E. Frank S.R. Maizels E.T. Bader M.-F. Kahn R.A. Palczewski K. Aktories K. Hunzicker-Dunn M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5901-5906Crossref PubMed Scopus (72) Google Scholar and present report). The inability of R280D ARNO to promote LH/CG receptor desensitization might be attributable to its inability to appropriately associate with the plasma membrane because of the mutation in its PH domain. A similar argument can be made for the inability of R280D to block desensitization in a dominant negative manner. Our results showing that R280D ARNO neither stimulates nor inhibits LH/CG receptor desensitization indirectly suggest that the PH domain of ARNO is indeed necessary at least for its membrane association. However, the ineffectiveness of the PI 3-kinase inhibitor wortmannin to block desensitization indicates that ARNO activation is not dependent on PIP3 generated by PI 3-kinase activation. As PIP3 does not appear to participate in the activation of ARNO leading to LH/CG receptor desensitization, it is likely that the predominant ARNO present in follicle membranes contains the triglycine motif in its PH domain, which does not selectively bind PIP3 but rather binds PIP2 with equivalent affinity (32Klarlund J. Tsiaras W. Holik J.J. Chawla A. Czech M.P. J. Biol. Chem. 2000; 275: 32816-32821Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 36Cullen P.J. Chardin P. Curr. Biol. 2000; 10: R876-R878Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). The recombinant E156K ARNO and ARNO proteins that we have used contain the triglycine motif and are regulated by PIP2 rather than PIP3 (13Santy L.C. Frank S.R. Hatfield J.C. Casanova J.E. Curr. Biol. 1999; 9: 1173-1176Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Our results therefore suggest that membrane PIP2, either locally synthesized or unmasked, is required for LH/CG receptor to activate ARNO. LH/CG receptor activation has been shown to promote PIP2 synthesis (37Dimino M.J. Snitzer J. Noland Jr., T.A. Biol. Reprod. 1987; 36: 97-102Crossref PubMed Scopus (9) Google Scholar, 38Davis J.S. Weakland L.L. Farese R.V. West L.A. J. Biol. Chem. 1987; 262: 8515-8521Abstract Full Text PDF PubMed Google Scholar). There is also evidence that ARF6 in a GTP- and phosphatidic acid-dependent manner can activate PI 4-phosphate 5-kinase (39Honda A. Nogami M. Yokozeki T. Yamazaki M. Nakamura H. Watanabe H. Kawamoto K. Nakayama K. Morris A.J. Frohman M.A. Kanaho Y. Cell. 1999; 99: 521-532Abstract Full Text Full Text PDF PubMed Scopus (699) Google Scholar) and PI 4-kinase (40Godi A. Pertile P. Meyers R. Marra P. DiTullio G. Iurisci C. Luini A. Corda D. De Matteis M.A. Nat. Cell Biol. 1999; 1: 280-287Crossref PubMed Scopus (451) Google Scholar) to increase PIP2 synthesis. Future studies are needed to determine how the ability of ARNO to activate ARF is regulated downstream of the LH/CG receptor. LH/CG receptor desensitization is also not inhibited by the βγ inhibitor peptide QEHA. The ineffectiveness of the βγ inhibitor peptide QEHA is supported by our earlier evidence that neither transducin βγ (at 1 μm) nor rat liver βγ (at 400 nm) stimulates LH/CG receptor desensitization and that the βγ scavenger GST-β-adrenergic receptor kinase-peptide fusion protein (at 14 μm) does not inhibit LH/CG receptor desensitization (41Rajagopalan-Gupta R.M. Mukherjee S. Zhu X. Ho Y.-K. Hamm H. Birnbaumer M. Birnbaumer L. Hunzicker-Dunn M. Endocrinology. 1999; 140: 1612-1621Crossref PubMed Scopus (16) Google Scholar). These results suggest that Gβγ binding to the PH domain of ARNO does not mediate LH/CG receptor activation of ARNO. These results, however, do not rule out the possibility that ARNO is anchored to the membrane via Gβγ proteins. In conclusion, these studies show that endogenousmembrane-delimited ARNO appears to be obligatory for the LH/CG receptor to promote β-arrestin1 release from its membrane docking site and receptor desensitization based on the ability of catalytically inactive ARNO to block both of these responses. Although ARNO activation by the LH/CG receptor is independent of PIP3 and Gβγ, ARNO activation could well be dependent on PIP2. The PH domain of ARNO also appears to be required for its membrane association, but the ligand that interacts with this domain to promote membrane binding is unlikely to be the same one that leads to its activation. We gratefully acknowledge the gifts of anti-arrestin antibody F4C1 from Dr. Larry Donoso of the Wills Eye Hospital Research Division, Philadelphia, PA, and QEHA peptide from Dr. Ravi Iyengar, Dept. of Pharmacology, Mount Sinai School of Medicine, City University of New York, NY. We also thank Dr. Evelyn Maizels for critically reading this manuscript and Dr. Subhendu Mukhopadhy for purifying recombinant E156K and R280D ARNO.

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