Attenuated PLD1 association and signalling at the H452Y polymorphic form of the 5-HT(2A) receptor.

The 5-HT2A receptor (5-HT2AR) is implicated in psychotropic changes within the central nervous system (CNS). A number of polymorphisms have been reported in the 5-HT2AR gene; one of these results in a non-synonymous change, H452Y, in the carboxy-terminal tail of the receptor protein. The minor allele (9% occurrence) has been statistically associated with CNS dysfunction such as impaired memory processing and resistance to neuroleptic treatment in schizophrenic patients. We investigated the impact of H452Y mutation of the 5-HT2AR expressed in COS7 cells on distinctly coupled intracellular signalling pathways from the receptor, focusing on the heterotrimeric G protein-independent phospholipase D (PLD) pathway, compared to the conventional Gq/11-linked phospholipase C (PLC) pathway. The H452Y mutation selectively attenuated PLD signalling, which as in the wild-type receptor, was mediated by a molecular complex involving PLD1 docked to the receptor's carboxy-terminal tail domain. Co-immunoprecipitation and GST-fusion protein experiments revealed that the H452Y mutation selectively reduced PLD1 binding to the receptor. Experiments with blocking peptides to mimic short sections of the 5-HT2AR tail sequence revealed that the peptide spanning residue 452 strongly reduced PLD but not PLC responses of the receptor. Similar observations were made when assessing both PLD responses and PLD-dependent cellular proliferation elicited by activation of 5-HT2ARs natively expressed in MCF-7 cells. Overall these findings indicate that the H452Y polymorphic variant of the 5-HT2AR displays selective disruption of its PLD signalling pathway. This may potentially play a role in the CNS dysfunction associated with the H452Y allele of the 5-HT2AR.

5-HT2A receptor signalling through phospholipase D1 associated with its C-terminal tail.

The 5-HT2AR (5-hydroxytryptamine-2A receptor) is a GPCR (G-protein-coupled receptor) that is implicated in the actions of hallucinogens and represents a major target of atypical antipsychotic agents. In addition to its classical signalling though PLC (phospholipase C), the receptor can activate several other pathways, including ARF (ADP-ribosylation factor)-dependent activation of PLD (phospholipase D), which appears to be achieved through a mechanism independent of heterotrimeric G-proteins. In the present study we show that wild-type and inactive constructs of PLD1 (but not PLD2) respectively facilitate and inhibit ARF-dependent PLD signalling by the 5-HT2AR. Furthermore we demonstrate that PLD1 specifically co-immunoprecipitates with the receptor and binds to a distal site in GST (glutathione transferase) fusion protein constructs of its C-terminal tail which is distinct from the ARF-interaction site, thereby suggesting the existence of a functional ARF-PLD signalling complex directly associated with this receptor. This reveals the spatial co-ordination of an important GPCR, transducer and effector into a physical complex that is likely to reinforce the impact of receptor activation on a heterotrimeric G-protein-independent signalling pathway. Signalling of this receptor through such non-canonical pathways may be important to its role in particular disorders.

Pituitary adenylate cyclase-activating polypeptide (PACAP)/PAC1HOP1 receptor activation coordinates multiple neurotrophic signaling pathways: Akt activation through phosphatidylinositol 3-kinase gamma and vesicle endocytosis for neuronal survival.

MAPK and Akt pathways are predominant mediators of trophic signaling for many neuronal systems. Among the vasoactive intestinal peptide/secretin/glucagon family of related peptides, pituitary adenylate cyclase-activating polypeptide (PACAP) binding to specific PAC(1) receptor isoforms can engage multiple signaling pathways and promote neuroprotection through mechanisms that are not well understood. Using a primary sympathetic neuronal system, the current studies demonstrate that PACAP activation of PAC(1)HOP1 receptors engages both MAPK and Akt neurotrophic pathways in an integrated program to facilitate neuronal survival after growth factor withdrawal. PACAP not only stimulated prosurvival ERK1/2 and ERK5 activation but also abrogated SAPK/JNK and p38 MAPK signaling in parallel. In contrast to the potent and rapid effects of PACAP in ERK1/2 phosphorylation, PACAP stimulated Akt phosphorylation in a late phase of PAC(1)HOP1 receptor signaling. From inhibitor and immunoprecipitation analyses, the PACAP/PAC(1)HOP1 receptor-mediated Akt responses did not represent transactivation mechanisms but appeared to depend on G alpha(q)/phosphatidylinositol 3-kinase gamma activity and vesicular internalization pathways. Phosphatidylinositol 3-kinase gamma-selective inhibitors blocked PACAP-stimulated Akt phosphorylation in primary neuronal cultures and in PAC(1)HOP1-overexpressing cell lines; RNA interference-mediated knockdown of the receptor effectors attenuated PACAP-mediated Akt activation. Similarly, perturbation of endocytic pathways also blocked Akt phosphorylation. Between ERK and Akt pathways, PACAP-stimulated Akt signaling was the primary cascade that attenuated cultured neuron apoptosis after growth factor withdrawal. The partitioning of PACAP-mediated Akt signaling in endosomes may be a key mechanism contributing to the high spatial and temporal specificity in signal transduction necessary for survival pathways.

Role of the conserved NPxxY motif of the 5-HT2A receptor in determining selective interaction with isoforms of ADP-ribosylation factor (ARF).

In this study we have shown that N376 to D mutation in the conserved NPxxY motif within the carboxy terminal tail domain (CT) of the 5-HT2A receptor alters the binding preference of GST-fusion protein constructs of the CT domain from ARF1 to an alternative isoform, ARF6. These findings were corroborated by experiments investigating co-immunoprecipitation of the wild type (WT) and N376D mutant of the 5-HT2A receptor with ARF1 or 6 or dominant negative ARF1/6 constructs co-expressed in COS7 cells. In functional assays of 5-HT-induced phospholipase D (PLD) activation responses of the WT receptor were inhibited by a dominant negative mutant of ARF1 but not ARF6, whereas responses of the N376D mutant were strongly inhibited by negative mutant ARF6. No equivalent effect of the ARF mutants was seen on phospholipase C activation. In experiments assaying 5-HT-induced increases in [35S]GTPgammaS binding to ARF 1/6 immunoprecipitates as a measure of ARF activation, increased ARF6 activation was seen only with the mutant receptor. When cellular PLD responses of other NPxxY- or a DPxxY-containing GPCRs were measured in the presence of dominant negative ARF1/6 constructs, the majority, but not all, fitted the pattern exemplified by the 5-HT2A receptor and its N376D mutant. These data suggest that the presence of the N or a D in this highly conserved motif is an important, but not exclusive, determinant of which ARF isoform interacts with the GPCR.

Location and function of VPAC1, VPAC2 and NPR-C receptors in VIP-induced vasodilation of porcine basilar arteries.

Vasoactive intestinal peptide (VIP) is a vasodilator peptide present in cerebrovascular nerves. Vasoactive intestinal peptide can activate VPAC1, VPAC2 and the NPR-C receptor. This study sought to determine the receptors involved in VIP-induced vasodilation of porcine basilar arteries. Porcine basilar arteries contained the messenger ribonucleic acid of all three receptors. Immunocytochemical analysis of porcine basilar arteries revealed that the VPAC1 receptor is expressed on the endothelium, VPAC2 on the outer layers of the media and the NPR-C receptor throughout the artery, including nerves. Vasodilator responses to all receptor agonists showed that the receptors are functional. The vasodilator response to the VPAC1 receptor agonist was inhibited by L-NAME and abolished by endothelial denudation. Vasodilation induced by Ro-25-1553, the VPAC2 agonist, was unaffected by NOS inhibition or removal of the endothelium. Activation of the NPR-C receptor produced a vasodilation, which was susceptible to NOS inhibition and independent of endothelium. The vasodilator response to electrical stimulation at 20 Hz was attenuated by PG-99-465, the VPAC2 antagonist. This study shows that all known VIP receptors are involved in VIP-mediated vasodilation of porcine basilar arteries. The VPAC1 receptor is located on the endothelium and elicits vasodilation by generating nitric oxide (NO). The VPAC2 receptor is mainly expressed in the outer layers of the smooth muscle and induces vasodilation independently of NO in response to VIP released from intramural nerves. The NPR-C receptor produces NO-dependent vasodilation independently of the endothelium by stimulation of nNOS in intramural nerves.

Selective interaction of ARF1 with the carboxy-terminal tail domain of the 5-HT2A receptor.

The 5-hydroxytryptamine 2A receptor (5-HT2AR) 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 Gq/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 Gq/11 (Mitchell et al., 1998). Both coimmunoprecipitation experiments and the effects of negative mutant ARF constructs on 5-HT2AR-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-HT2AR. 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 GTPgammaS 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-HT2AR 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.

ADP-ribosylation factor-dependent phospholipase D activation by the M3 muscarinic receptor.

G protein-coupled receptors can potentially activate phospholipase D (PLD) by a number of routes. We show here that the native M3 muscarinic receptor in 1321N1 cells and an epitope-tagged M3 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, phospholipase C, Ca2+ q/11, protein kinase C, tyrosine kinases, and phosphatidyl inositol 3-kinase. We report instead that it involves physical association of ARF with the M3 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 M3 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.

Functional interactions between native Gs-coupled 5-HT receptors in HEK-293 cells and the heterologously expressed serotonin transporter.

In HEK-293 cells, serotonin (5-hydroxytryptamine, 5-HT) was found to induce cAMP production showing pharmacological characteristics consistent with the 5-HT(7) receptor. The presence of 5-HT(7) (and 5-HT(6)) receptor mRNA was confirmed by RT-PCR. Stable HEK-293 cell lines expressing either wild-type or haemagglutinin (HA)-tagged human 5-HT transporter (SERT) were selected and SERT function was confirmed using [3H]5-HT transport. The presence of SERT caused a 10-fold reduction in the potency of 5-HT-induced cAMP production compared to control cells. Downstream signalling by 5-HT(6/7) receptors could be detected as 5-HT-induced protein kinase A activation and phosphorylation of MAP kinase and CREB using phospho-specific antibodies. SERT inhibitors reversed the reduction in potency of 5-HT-induced cAMP production caused by the presence of SERT, resulting in a concentration-dependent left shift in EC(50) values but also a progressive decrease in the maximal response. Thus, when antidepressants were used to block SERT activity, 5-HT receptor signalling was effectively clamped within a mid-range.