G protein-coupled receptors can potentially activate phospholipase D (PLD) by a number of routes. We show here that the native M 3 muscarinic receptor in 1321N1 cells and an epitope-tagged M 3 receptor expressed in COS7 cells substantially utilize an ADP-ribosylation factor (ARF)-dependent route of PLD activation. This pathway is activated at the plasma membrane but appears to be largely independent of G q/11 , phospholipase C, Ca 2؉ , protein kinase C, tyrosine kinases, and phosphatidyl inositol 3-kinase. We report instead that it involves physical association of ARF with the M 3 receptor as demonstrated by co-immunoprecipitation and by in vitro interaction with a glutathione S-transferase fusion protein of the receptor's third intracellular loop domain. Experiments with mutant constructs of ARF1/6 and PLD1/2 indicate that the M 3 receptor displays a major ARF1-dependent route of PLD1 activation with an additional ARF6-dependent pathway to PLD1 or PLD2. Examples of other G protein-coupled receptors assessed in comparison display alternative pathways of protein kinase C-or ARF6-dependent activation of PLD2. Many G protein-coupled receptors (GPCRs)1 can activate phospholipase D (PLD), which catalyzes the hydrolysis of phosphatidylcholine to phosphatidic acid and choline. Both phosphatidates and diacylglycerols (formed by phosphatidate hydrolysis) may act as intracellular messengers. PLD has been implicated as a key regulator of vesicular trafficking, cytoskeletal organization, exocytosis, endocytosis, and further signaling pathways (1-4). Activation of PLD can be brought about by a variety of signaling events (5-8), many of which could potentially contribute to the stimulation of PLD activity by GPCRs. These include the activation of protein kinase C (PKC), proteintyrosine kinases, phosphatidylinositol 3-kinase (PI 3-kinase), small G proteins of the ARF and Rho families, and possibly the elevation of intracellular Ca 2ϩ levels. This study addresses the mechanism of PLD activation by the M 3 muscarinic receptor expressed endogenously in 1321N1 human astrocytoma cells and heterologously in COS7 cells. The M 3 receptor is a member of the Group I, rhodopsin-related GPCR family that is expressed in the nervous system and peripheral tissues. The best established signaling pathway from the M 3 receptor is the pertussis toxin-insensitive activation of phospholipase C (PLC) via the heterotrimeric G protein G q/11 , although PLD is also strongly activated. In various cell types, PKC, protein-tyrosine kinases, ARF, and Rho have each been specifically implicated in M 3 receptor-mediated PLD activation (6, 9 -12). The data here emphasize the importance of a pathway to PLD that involves direct association between ARF and the M 3 receptor (12).ARF1 and ARF6 are representative of the main classes of cellular ARFs (Classes I and III) and have distinct subcellular distributions in many cell types. In resting cells, ARF1 is largely cytosolic or Golgi-associated, whereas ARF6 is often localized to the plasma membrane (13-17). Nevert...

The 5-hydroxytryptamine 2A receptor (5-HT 2A R) is a member of the class I family of rhodopsin-related G protein-coupled receptors. The receptor is known to activate phospholipase C via the heterotrimeric G proteins G q/11 , but we showed previously that it can also signal through the phospholipase D (PLD) pathway in an ADP-ribosylation factor (ARF)-dependent manner that seems to be independent of G q/11 (Mitchell et al., 1998). Both coimmunoprecipitation experiments and the effects of negative mutant ARF constructs on 5-HT 2A R-induced PLD activation here suggested that ARF1 may play a greater role than ARF6 in the function of this receptor. Furthermore, we demonstrated using glutathione S-transferase (GST)-fusion proteins of receptor domains that ARF1 and ARF6 bind to the third intracellular loop (i3) and the carboxy terminal tail (ct) of the 5-HT 2A R. The association of ARF1 with the ct domain of the receptor was stronger than its interaction with i3, or the interactions of ARF6 with either construct. Experiments using ARF mutants that are deficient in GTP loading, and the in vitro addition of GTP␥S suggested that GTP loading enhances ARF1 binding to the receptor. The N376PxxY motif in the transmembrane 7 domain of the receptor (rather than a N376DPxxY mutant form) was shown to be essential for ARF-dependent PLD signaling and ARF1 coimmunoprecipitation. In GST-fusion proteins of the 5-HT 2A R ct domain, mutation of Asn376 to Asp also markedly reduced ARF1-HA binding, although additional motifs in the Asn376 -Asn384 sequence and to a lesser extent elsewhere, seem also to contribute to the interaction.

Structural microdomains of G protein-coupled receptors (GPCRs) consist of spatially related side chains that mediate discrete functions. The conserved helix 2/helix 7 microdomain was identified because the gonadotropin-releasing hormone (GnRH) receptor appears to have interchanged the Asp 2.50 and Asn 7.49 residues which are conserved in transmembrane helices 2 and 7 of rhodopsin-like GPCRs. We now demonstrate that different side chains of this microdomain contribute specifically to receptor expression, heterotrimeric G protein-, and small G protein-mediated signaling. An Asn residue is required in position 2.50(87) for expression of the GnRH receptor at the cell surface, most likely through an interaction with the conserved Asn 1.50 (53) residue, which we also find is required for receptor expression. Most GPCRs require an Asp side chain at either the helix 2 or helix 7 locus of the microdomain for coupling to heterotrimeric G proteins, but the GnRH receptor has transferred the requirement for an acidic residue from helix 2 to 7. However, the presence of Asp at the helix 7 locus precludes small G protein-dependent coupling to phospholipase D. These results implicate specific components of the helix 2/helix 7 microdomain in receptor expression and in determining the ability of the receptor to adopt distinct activated conformations that are optimal for interaction with heterotrimeric and small G proteins.The gonadotropin-releasing hormone (GnRH) 1 receptor belongs to the rhodopsin-like family of G protein-coupled receptors (GPCR) (1). This family includes the light-sensitive opsins, protease-activated receptors, and receptors for neurotransmitters, peptides, and glycoproteins. High resolution structural data have not yet been obtained for any GPCR. However, projection maps of rhodopsin, amino acid sequence alignment, and computational modeling indicate that GPCRs have 7 membrane-spanning ␣-helices (2-6). There is a high degree of homology within the transmembrane helices and certain amino acids are highly conserved throughout the family (2, 3, 7). This diverse family shares the common function of propagating a signal across lipid membranes and the amino acid side chains which are conserved among the GPCRs are likely to constitute key structural motifs which subserve this universal GPCR function. Several models of GPCRs, including the GnRH receptor (4, 8), have been constructed as aids for investigating receptor structure-function relations. Molecular models of GPCRs can be used to integrate experimental observations and generate structural hypotheses. However, the complexity of these structures and the limited number of experimentally determined constraints can lead to inconsistent behavior of the models (4, 7). To overcome these limitations, we have pursued the approach of identifying discrete structural motifs within receptor models, which might constitute functional microdomains. The microdomains are characterized in detail and subsequently incorporated into whole receptor models. In the GnRH receptor, for exa...

The VPAC(1) and VPAC(2) receptors for vasoactive intestinal polypeptide and the PAC(1) receptor for pituitary adenylate cyclase-activating polypeptide are members of a subfamily of G protein-coupled receptors (GPCRs). We recently reported that phospholipase D (PLD) activation by members of the rhodopsin group of GPCRs occurs by at least two routes, one of which seems to involve the small G protein ADP-ribosylation factor (ARF) and its physical association with GPCRs. Here we report that rat VPAC and PAC(1) receptors can also stimulate PLD (albeit less potently than adenylate cyclase) in transfected cells and also in cells where they are natively expressed. PLD responses of the VPAC receptors and the hop1 spice variant of the PAC(1) receptor but not its null form are sensitive to brefeldin A (BFA), an inhibitor of GTP exchange at ARF. The presence of the hop1 cassette in the rat PAC(1) receptor facilitates PLD activation in the absence of marked changes in ligand binding, receptor internalization, and adenylate cyclase activation, with some reduction in phospholipase C activation. Both VPAC(2) and PAC(1-hop1) (but not PAC(1-null)) receptors were shown to associate with immunoprecipitates directed against native or epitope-tagged ARF. A chimeric construct of the VPAC(2) receptor body with intracellular loop 3 (i3) of the PAC(1-null) receptor mediated BFA-insensitive activation of PLD, whereas the response of the corresponding PAC(1-hop1) construct was BFA-sensitive. Motifs in i3 of the PAC(1-hop1) receptor may act as critical determinants of coupling to ARF-dependent PLD activation by contributing to the GPCR:ARF interface.