The 5-hydroxytryptamine(4a) receptor is palmitoylated at two different sites, and acylation is critically involved in regulation of receptor constitutive activity.

We have reported recently that the mouse 5-hydroxytryptamine(4a) (5-HT(4(a))) receptor undergoes dynamic palmitoylation (Ponimaskin, E. G., Schmidt, M. F., Heine, M., Bickmeyer, U., and Richter, D. W. (2001) Biochem. J. 353, 627-663). In the present study, conserved cysteine residues 328/329 in the carboxyl terminus of the 5-HT(4(a)) receptor were identified as potential acylation sites. In contrast to other palmitoylated G-protein-coupled receptors, the additional cysteine residue 386 positioned close to the COOH-terminal end of the receptor was also found to be palmitoylated. Using pulse and pulse-chase labeling techniques, we demonstrated that palmitoylation of individual cysteines is a reversible process and that agonist stimulation of the 5-HT(4(a)) receptor independently increases the rate of palmitate turnover for both acylation sites. Analysis of acylation-deficient mutants revealed that non-palmitoylated 5-HT(4(a)) receptors were indistinguishable from the wild type in their ability to interact with G(s), to stimulate the adenylyl cyclase activity and to activate cyclic nucleotide-sensitive cation channels after agonist stimulation. The most distinctive finding of the present study was the ability of palmitoylation to modulate the agonist-independent constitutive 5-HT(4(a)) receptor activity. We demonstrated that mutation of the proximal palmitoylation site (Cys(328) --> Ser/Cys(329) --> Ser) significantly increases the capacity of receptors to convert from the inactive (R) to the active (R*) form in the absence of agonist. In contrast, the rate of isomerization from R to R* for the Cys(386) --> Ser as well as for the triple, non-palmitoylated mutant (Cys(328) --> Ser/Cys(329) --> Ser/Cys(386) -->Ser) was similar to that obtained for the wild type.     

5-hydroxytryptamine 4 receptor activation of the extracellular signal-regulated kinase pathway depends on Src activation but not on G protein or beta-arrestin signaling

The 5-hydroxytryptamine(4) (5-HT(4)) receptors have recently emerged as key modulators of learning, memory, and cognitive processes. In neurons, 5-hydroxytryptamine(4) receptors (5-HT(4)Rs) activate cAMP production and protein kinase A (PKA); however, nothing is known about their ability to activate another key signaling pathway involved in learning and memory: the extracellular signal-regulated kinase (ERK) pathway. Here, we show that 5-HT(4)R stimulation, in primary neurons, produced a potent but transient activation of the ERK pathway. Surprisingly, this activation was mostly PKA independent. Similarly, using pharmacological, genetic, and molecular tools, we observed that 5-HT(4)Rs in human embryonic kidney 293 cells, activated the ERK pathway in a G(s)/cAMP/PKA-independent manner. We also demonstrated that other classical G proteins (G(q)/G(i)/G(o)) and associated downstream messengers were not implicated in the 5-HT(4)R-activated ERK pathway. The 5-HT(4)R-mediated ERK activation seemed to be dependent on Src tyrosine kinase and yet totally independent of beta-arrestin. Immunocytofluorescence revealed that ERK activation by 5-HT(4)R was restrained to the plasma membrane, whereas p-Src colocalized with the receptor and carried on even after endocytosis. This phenomenon may result from a tight interaction between 5-HT(4)R and p-Src detected by coimmunoprecipitation. Finally, we confirmed that the main route by which 5-HT(4)Rs activate ERKs in neurons was Src dependent. Thus, in addition to classical cAMP/PKA signaling pathways, 5-HT(4)Rs may use ERK pathways to control memory process.     

Endogenous RGS protein action modulates mu-opioid signaling through Galphao. Effects on adenylyl cyclase,extracellular signal-regulated kinases,and intracellular calcium pathways

RGS (regulators of G protein signaling) proteins are GTPase-activating proteins for the Galpha subunits of heterotrimeric G proteins and act to regulate signaling by rapidly cycling G protein. RGS proteins may integrate receptors and signaling pathways by physical or kinetic scaffolding mechanisms. To determine whether this results in enhancement and/or selectivity of agonist signaling, we have prepared C6 cells stably expressing the mu-opioid receptor and either pertussis toxin-insensitive or RGS- and pertussis toxin-insensitive Galpha(o). We have compared the activation of G protein, inhibition of adenylyl cyclase, stimulation of intracellular calcium release, and activation of the ERK1/2 MAPK pathway between cells expressing mutant Galpha(o) that is either RGS-insensitive or RGS-sensitive. The mu-receptor agonist [d-Ala(2),MePhe(4),Gly(5)-ol]enkephalin and partial agonist morphine were much more potent and/or had an increased maximal effect in inhibiting adenylyl cyclase and in activating MAPK in cells expressing RGS-insensitive Galpha(o). In contrast, mu-opioid agonist increases in intracellular calcium were less affected. The results are consistent with the hypothesis that the GTPase-activating protein activity of RGS proteins provides a control that limits agonist action through effector pathways and may contribute to selectivity of activation of intracellular signaling pathways.     

Molecular analysis of the interaction between the intracellular loops of the human serotonin receptor type 6 (5-HT6) and the alpha subunit of GS protein

The serotonin type 6 (5-HT(6)) receptor is a G-protein coupled receptor (GPCR) coupled to a stimulatory G-protein (G(S)). To identify the structural basis for the interaction of the 5-HT(6) receptor with the G(S) protein, we have dissected the interaction between GST-fusion proteins containing the second intracellular loop (iL2), the third intracellular loop (iL3), or the C-terminal tail of the 5-HT(6) receptor and the alpha subunit of G(S) (Galpha(S)). The direct interaction of iL3 and Galpha(S) was demonstrated by co-immunoprecipitation. Furthermore, the kinetic parameters of the interaction between iL3 and Galpha(S) were measured by surface plasmon resonance, and the apparent dissociation constant was determined to be 0.9 x 10(-6)M. In contrast, the second intracellular loop and C-terminal tail regions showed negligible affinity to Galpha(S). The critical residues within the iL3 region for the interaction with Galpha(S) were identified as conserved positively charged residues near the C-terminus of iL3 by measuring the cellular levels of cAMP produced in response to 5-HT stimulation of cells transfected with 5-HT(6) receptor mutants.     

Two amino acids within the alpha4 helix of Galphai1 mediate coupling with 5-hydroxytryptamine1B receptors.

We previously reported that residues 299-318 in Galphai1 participate in the selective interaction between Galphai1 and the 5-hydroxytryptamine1B (5-HT1B) receptor (Bae, H., Anderson, K., Flood, L. A., Skiba, N. P., Hamm, H. E., and Graber, S. G. (1997) J. Biol. Chem. 272, 32071-32077). The present study more precisely defines which residues within this domain are critical for 5-HT1B receptor-mediated G protein activation. A series of Galphai1/Galphat chimeras and point mutations were reconstituted with Gbetagamma and Sf9 cell membranes containing the 5-HT1B receptor. Functional coupling to 5-HT1B receptors was assessed by 1) [35S]GTPgammaS binding and 2) agonist affinity shift assays. Replacement of the alpha4 helix of Galphai1 (residues 299-308) with the corresponding sequence from Galphat produced a chimera (Chi22) that only weakly coupled to the 5-HT1B receptor. In contrast, substitution of residues within the alpha4-beta6 loop region of Galphai1 (residues 309-318) with the corresponding sequence in Galphat either permitted full 5-HT1B receptor coupling to the chimera (Chi24) or only minimally reduced coupling to the chimeric protein (Chi25). Two mutations within the alpha4 helix of Galphai1 (Q304K and E308L) reduced agonist-stimulated [35S]GTPgammaS binding, and the effects of these mutations were additive. The opposite substitutions within Chi22 (K300Q and L304E) restored 5-HT1B receptor coupling, and again the effects of the two mutations were additive. Mutations of other residues within the alpha4 helix of Galphai1 had minimal to no effect on 5-HT1B coupling behavior. These data provide evidence that alpha4 helix residues in Galphai participate in directing specific receptor interactions and suggest that Gln304 and Glu308 of Galphai1 act in concert to mediate the ability of the 5-HT1B receptor to couple specifically to inhibitory G proteins.     

Differential involvement of Galpha12 and Galpha13 in receptor-mediated stress fiber formation.

The ubiquitously expressed heterotrimeric guanine nucleotide-binding proteins (G-proteins) G12 and G13 have been shown to activate the small GTPase Rho. Rho stimulation leads to a rapid remodeling of the actin cytoskeleton and subsequent stress fiber formation. We investigated the involvement of G12 or G13 in stress fiber formation induced through a variety of Gq/G11-coupled receptors. Using fibroblast cell lines derived from wild-type and Galphaq/Galpha11-deficient mice, we show that agonist-dependent activation of the endogenous receptors for thrombin or lysophosphatidic acid and of the heterologously expressed bradykinin B2, vasopressin V1A, endothelin ETA, and serotonin 5-HT2C receptors induced stress fiber formation in either the presence or absence of Galphaq/Galpha11. Stress fiber assembly induced through the muscarinic M1 and the metabotropic glutamate subtype 1alpha receptors was dependent on Gq/G11 proteins. The activation of the Gq/G11-coupled endothelin ETB and angiotensin AT1A receptors failed to induce stress fiber formation. Lysophosphatidic acid, B2, and 5-HT2C receptor-mediated stress fiber formation was dependent on Galpha13 and involved epidermal growth factor (EGF) receptors, whereas thrombin, ETA, and V1A receptors induced stress fiber accumulation via Galpha12 in an EGF receptor-independent manner. Our data demonstrate that many Gq/G11-coupled receptors induce stress fiber assembly in the absence of Galphaq and Galpha11 and that this involves either a Galpha12 or a Galpha13/EGF receptor-mediated pathway.     

The 5-hydroxytryptamine(1A) receptor is stably palmitoylated, and acylation is critical for communication of receptor with Gi protein

In the present study, we verified that the mouse 5-hydroxytryptamine(1A) (5-HT(1A)) receptor is modified by palmitic acid, which is covalently attached to the protein through a thioester-type bond. Palmitoylation efficiency was not modulated by receptor stimulation with agonists. Block of protein synthesis by cycloheximide resulted in a significant reduction of receptor acylation, suggesting that palmitoylation occurs early after synthesis of the 5-HT(1A) receptor. Furthermore, pulse-chase experiments demonstrated that fatty acids are stably attached to the receptor. Two conserved cysteine residues 417 and 420 located in the proximal C-terminal domain were identified as acylation sites by site-directed mutagenesis. To address the functional role of 5-HT(1A) receptor acylation, we have analyzed the ability of acylation-deficient mutants to interact with heterotrimeric G(i) protein and to modulate downstream effectors. Replacement of individual cysteine residues (417 or 420) resulted in a significantly reduced coupling of receptor with G(i) protein and impaired inhibition of adenylyl cyclase activity. When both palmitoylated cysteines were replaced, the communication of receptors with G alpha(i) subunits was completely abolished. Moreover, non-palmitoylated mutants were no longer able to inhibit forskolin-stimulated cAMP formation, indicating that palmitoylation of the 5-HT(1A) receptor is critical for the enabling of G(i) protein coupling/effector signaling. The receptor-dependent activation of extracellular signal-regulated kinase was also affected by acylation-deficient mutants, suggesting the importance of receptor palmitoylation for the signaling through the G beta gamma-mediated pathway, in addition to the G alpha(i)-mediated signaling.     

Cell-specific signaling of the 5-HT1A receptor. Modulation by protein kinases C and A.

Heterologous expression of the rat 5-HT1A receptor in stably transfected GH4C1 rat pituitary cells (clone GH4ZD10) and mouse Ltk- fibroblast cells (clone LZD-7) (Albert, P.R., Zhou, Q.-Y., VanTol, H.H.M., Bunzow, J.R., and Civelli, O. (1990) J. Biol. Chem. 265, 5825-5832) was used to characterize the cellular specificity of signal transduction by the 5-HT1A receptor. We demonstrate that the 5-HT1A receptor, acting via pertussis toxin-sensitive G proteins, can change its inhibitory signaling phenotype and become a stimulatory receptor, depending on the cell type, differentiation state, or intracellular milieu of the cell in which it is expressed. When expressed in pituitary GH4ZD10 cells, activation of 5-HT1A receptors decreased both basal and vasoactive intestinal peptide-enhanced cAMP accumulation and blocked (+/-)-Bay K8644-induced influx of calcium, inhibitory responses which are typical of neurons which endogenously express this receptor. Similarly, 5-hydroxytryptamine (5-HT) also inhibited adenylyl cyclase in fibroblast LZD-7 cells, reducing the forskolin-induced enhancement of cAMP levels by 50%, but did not alter basal cAMP levels. In contrast to GH4ZD10 cells, where 5-HT had no effect on basal or thyrotropin-releasing hormone-induced phosphatidylinositol turnover, 5-HT enhanced the accumulation of inositol phosphates and induced a biphasic increase in [Ca2+]i in LZD-7 cells. These dominant stimulatory actions of 5-HT, as well as the inhibitory effects, were absent in untransfected cells and displayed the potency and pharmacological specificity of the 5-HT1A receptor, indicating that the 5-HT1A subtype coupled to both inhibitory and stimulatory pathways in the fibroblast cell. The actions of 5-HT in GH and L cells were blocked by 24-h pretreatment with pertussis toxin, suggesting that inhibitory G proteins (Gi/G(o)) mediate both inhibitory and stimulatory signal transduction of the 5-HT1A receptor. However, the 5-HT-induced stimulatory pathway in fibroblasts was blocked selectively by acute (2-min) pretreatment with TPA, an activator of protein kinase C. This action of protein kinase C was potentiated by activation of protein kinase A, indicating that the expression of the stimulatory pathway of the 5-HT1A receptor in LZD-7 cells is modulated by second messengers.     

The 5-HT3B subunit confers spontaneous channel opening and altered ligand properties of the 5-HT3 receptor

Current receptor theory suggests that there is an equilibrium between the inactive (R) and active (R*) conformations of ligand-gated ion channels and G protein-coupled receptors. The actions of ligands in both receptor types could be appropriately explained by this two-state model. Ligands such as agonists and antagonists affect receptor function by stabilizing one or both conformations. The 5-HT3 receptor is a member of the Cys-loop ligand-gated ion channel superfamily participating in synaptic transmission. Here we show that co-expression of the 5-HT3A and 5-HT3B receptor subunits in the human embryonic kidney (HEK) 293 cells results in a receptor that displays a low level of constitutive (or agonist-independent) activity. Furthermore, we also demonstrate that the properties of ligands can be modified by receptor composition. Whereas the 5-hydroxytryptamine (5-HT) analog 5-methoxyindole is a partial agonist at the 5-HT3A receptor, it becomes a "protean agonist" (functioning as an agonist and an inverse agonist at the same receptor) at the 5-HT3AB receptor (after the Greek god Proteus, who was able to change his shape and appearance at will). In addition, the 5-HT analog 5-hydroxyindole is a positive allosteric modulator for the liganded active (AR*) conformation of the 5-HT3A and 5-HT3AB receptors and a negative allosteric modulator for the spontaneously active (R*) conformation of the 5-HT3AB receptor, suggesting that the spontaneously active (R*) and liganded active (AR*) conformations are differentially modulated by 5-hydroxyindole. Thus, the incorporation of the 5-HT3B subunit leads to spontaneous channel opening and altered ligand properties.     

Dissecting G protein-coupled receptor signaling pathways with membrane-permeable blocking peptides. Endogenous 5-HT(2C) receptors in choroid plexus epithelial cells

To determine the intracellular signaling mechanism of the 5-HT(2C) receptor endogenously expressed in choroid plexus epithelial cells, we implemented a strategy of targeted disruption of protein-protein interactions. This strategy entails the delivery of conjugated membrane-permeable peptides that disrupt domain interaction at specific steps in the signaling cascade. As proof of concept, two peptides targeted against receptor-G protein interaction domains were examined. Only G(q)CT, which targets the receptor-G(q) protein interacting domain, disrupted 5-HT(2C) receptor-mediated phosphatidylinositide hydrolysis. G(s)CT, targeting the receptor-G(s) protein, disrupted beta2 adrenergic receptor-mediated activation of cAMP but not 5-HT(2C) receptor-mediated phosphatidylinositide hydrolysis. The peptide MPS-PLCbeta1M, mimicking the domain of phospholipase Cbeta1 (PLCbeta1) interacting with active Galpha(q), also blocked 5-HT(2C) receptor activation. In contrast, peptides PLCbeta2M and Phos that bind to and sequester free Gbetagamma subunits were ineffective at blocking 5-HT(2C) receptor-mediated phosphoinositol turnover. However, both peptides disrupted Gbetagamma-mediated alpha(2A) adrenergic receptor activation of mitogen-activated protein kinase. These results provide the first direct demonstration that active Galpha(q) subunits mediate endogenous 5-HT(2C) receptor activation of PLCbeta and that Gbetagamma subunits released from Galpha(q) heterotrimeric proteins are not involved. Comparable results were obtained with metabotropic glutamate receptor 5 expressed in astrocytes. Thus, conjugated, membrane-permeable peptides are effective tools for the dissection of intracellular signals.     

Constitutive G(i2)-dependent activation of adenylyl cyclase type II by the 5-HT1A receptor. Inhibition by anxiolytic partial agonists

The 5-HT1A receptor is implicated in depression and anxiety. This receptor couples to G(i) proteins to inhibit adenylyl cyclase (AC) activity but can stimulate AC in tissues (e.g. hippocampus) that express ACII. The role of ACII in receptor-mediated stimulation of cAMP formation was examined in HEK-293 cells transfected with the 5-HT1A receptor, which mediated inhibition of basal and G(s)-induced cAMP formation in the absence of ACII. In cells cotransfected with 5-HT1A receptor and ACII plasmids, 5-HT1A agonists induced a 1. 5-fold increase in cAMP level. Cotransfection of 5-HT1A receptor, ACII, and Galpha(i2), but not Galpha(i1), Galpha(i3), or Galpha(o), resulted in an agonist-independent 6-fold increase in the basal cAMP level, suggesting that G(i2) preferentially coupled the receptor to ACII. The 5-HT1B receptor also constitutively activated ACII. Constitutive activity of the 5-HT1A receptor was blocked by pertussis toxin and the Gbetagamma antagonist, betaCT, suggesting an important role for Gbetagamma-mediated activation of ACII. The Thr-149 --> Ala mutation in the second intracellular domain of the 5-HT1A receptor disrupted Gbetagamma-selective activation of ACII. Spontaneous 5-HT1A receptor activity was partially attenuated by 5-HT1A receptor partial agonists with anxiolytic activity (e.g. buspirone and flesinoxan) but was not altered by full agonists or antagonists. Thus, anxiolytic activity may involve inhibition of spontaneous 5-HT1A receptor activity.     

The Galpha13-Rho signaling axis is required for SDF-1-induced migration through CXCR4

The CXC chemokine stromal cell-derived factor-1alpha (SDF-1) binds to CXCR4, a seven-transmembrane G protein-coupled receptor that plays a critical role in many physiological processes that involve cell migration and cell fate decisions, ranging from stem cell homing, angiogenesis, and neuronal development to immune cell trafficking. CXCR4 is also implicated in various pathological conditions, including metastatic spread and human immunodeficiency virus infection. Although SDF-1-induced cell migration in CXCR4-expressing cells is sensitive to pertussis toxin treatment, hence involving heterotrimeric G proteins of the G(i) family, whether other G proteins participate in the chemotactic response to SDF-1 is still unknown. In this study, we took advantage of the potent chemotactic activity of SDF-1 in Jurkat T-cells to examine the nature of the heterotrimeric G protein subunits contributing to CXCR4-mediated cell migration. We observed that whereas G(i) and Gbetagamma subunits are involved in SDF-1-induced Rac activation and cell migration, CXCR4 can also stimulate Rho potently leading to the phosphorylation of myosin light chain through the Rho effector, Rho kinase, but independently of G(i). Furthermore, we found that Galpha(13) mediates the activation of Rho by CXCR4 and that the functional activity of both Galpha(13) and Rho is required for directional cell migration in response to SDF-1. Collectively, our data indicate that signaling by CXCR4 to Rho through Galpha(13) contributes to cell migration when stimulated by SDF-1, thus identifying the Galpha(13)-Rho signaling axis as a potential pharmacological target in many human diseases that involve the aberrant function of CXCR4.     

Selective regulation of Galpha(q/11) by an RGS domain in the G protein-coupled receptor kinase, GRK2

G protein-coupled receptor kinases (GRKs) are well characterized regulators of G protein-coupled receptors, whereas regulators of G protein signaling (RGS) proteins directly control the activity of G protein alpha subunits. Interestingly, a recent report (Siderovski, D. P., Hessel, A., Chung, S., Mak, T. W., and Tyers, M. (1996) Curr. Biol. 6, 211-212) identified a region within the N terminus of GRKs that contained homology to RGS domains. Given that RGS domains demonstrate AlF(4)(-)-dependent binding to G protein alpha subunits, we tested the ability of G proteins from a crude bovine brain extract to bind to GRK affinity columns in the absence or presence of AlF(4)(-). This revealed the specific ability of bovine brain Galpha(q/11) to bind to both GRK2 and GRK3 in an AlF(4)(-)-dependent manner. In contrast, Galpha(s), Galpha(i), and Galpha(12/13) did not bind to GRK2 or GRK3 despite their presence in the extract. Additional studies revealed that bovine brain Galpha(q/11) could also bind to an N-terminal construct of GRK2, while no binding of Galpha(q/11), Galpha(s), Galpha(i), or Galpha(12/13) to comparable constructs of GRK5 or GRK6 was observed. Experiments using purified Galpha(q) revealed significant binding of both Galpha(q) GDP/AlF(4)(-) and Galpha(q)(GTPgammaS), but not Galpha(q)(GDP), to GRK2. Activation-dependent binding was also observed in both COS-1 and HEK293 cells as GRK2 significantly co-immunoprecipitated constitutively active Galpha(q)(R183C) but not wild type Galpha(q). In vitro analysis revealed that GRK2 possesses weak GAP activity toward Galpha(q) that is dependent on the presence of a G protein-coupled receptor. However, GRK2 effectively inhibited Galpha(q)-mediated activation of phospholipase C-beta both in vitro and in cells, possibly through sequestration of activated Galpha(q). These data suggest that a subfamily of the GRKs may be bifunctional regulators of G protein-coupled receptor signaling operating directly on both receptors and G proteins.     

5-HT induces duodenal mucosal bicarbonate secretion via cAMP- and Ca2+-dependent signaling pathways and 5-HT4 receptors in mice

In previous studies, we have found that 5-hydroxytryptamine (5-HT) is a potent stimulant of duodenal mucosal bicarbonate secretion (DMBS) in mice. The aim of the present study was to determine the intracellular signaling pathways and 5-HT receptor subtypes involved in 5-HT-induced DMBS. Bicarbonate secretion by murine duodenal mucosa was examined in vitro in Ussing chambers. 5-HT receptor involvement in DMBS was inferred from pharmacological studies by using selective 5-HT receptor antagonists and agonists. The expression of 5-HT(4) receptor mRNA in duodenal mucosa and epithelial cells was analyzed by RT-PCR. cAMP-dependent signaling pathway inhibitors MDL-12330A, Rp-cAMP, and H-89 and Ca(2+)-dependent signaling pathway inhibitors verapamil and W-13 markedly reduced 5-HT-stimulated duodenal bicarbonate secretion and short-circuit current (I(sc)), whereas cGMP-dependent signaling pathway inhibitors NS-2028 and KT-5823 failed to alter these responses. Both SB-204070 and high-dose ICS-205930 (selective 5-HT(4) receptor antagonists) markedly inhibited 5-HT-stimulated bicarbonate secretion and I(sc), whereas methiothepine (5-HT(1) receptor antagonist), ketanserin (5-HT(2) receptor antagonist), and a low concentration of ICS-205930 (5-HT(3) receptor antagonist) had no effect. RS-67506 (partial 5-HT(4) receptor agonist) concentration-dependently increased bicarbonate secretion and I(sc), whereas 5-carboxamidotryptamine (5-HT(1) receptor agonist), alpha-methyl-5-HT (5-HT(2) receptor agonist), and phenylbiguanide (5-HT(3) receptor agonist) did not significantly increase bicarbonate secretion or I(sc). RT-PCR analysis confirmed the expression of 5-HT(4) receptor mRNA in murine duodenal mucosa and epithelial cells. These results demonstrate that 5-HT regulates DMBS via both cAMP- and Ca(2+)-dependent signaling pathways and 5-HT(4) receptors located in the duodenal mucosa and/or epithelial cells.     

G protein activation by serotonin type 4 receptor dimers: evidence that turning on two protomers is more efficient

The discovery that class C G protein-coupled receptors (GPCRs) function as obligatory dimeric entities has generated major interest in GPCR oligomerization. Oligomerization now appears to be a common feature among all GPCR classes. However, the functional significance of this process remains unclear because, in vitro, some monomeric GPCRs, such as rhodopsin and β(2)-adrenergic receptors, activate G proteins. By using wild type and mutant serotonin type 4 receptors (5-HT(4)Rs) (including a 5-HT(4)-RASSL) expressed in COS-7 cells as models of class A GPCRs, we show that activation of one protomer in a dimer was sufficient to stimulate G proteins. However, coupling efficiency was 2 times higher when both protomers were activated. Expression of combinations of 5-HT(4), in which both protomers were able to bind to agonists but only one could couple to G proteins, suggested that upon agonist occupancy, protomers did not independently couple to G proteins but rather that only one G protein was activated. Coupling of a single heterotrimeric G(s) protein to a receptor dimer was further confirmed in vitro, using the purified recombinant WT RASSL 5-HT(4)R obligatory heterodimer. These results, together with previous findings, demonstrate that, differently from class C GPCR dimers, class A GPCR dimers have pleiotropic activation mechanisms.     

Constitutively active Gq/11-coupled receptors enable signaling by co-expressed G(i/o)-coupled receptors

Co-expression of guanine nucleotide-binding regulatory (G) protein-coupled receptors (GPCRs), such as the G(i/o)-coupled human 5-hydroxytryptamine receptor 1B (5-HT(1B)R), with the G(q/11)-coupled human histamine 1 receptor (H1R) results in an overall increase in agonist-independent signaling, which can be augmented by 5-HT(1B)R agonists and inhibited by a selective inverse 5-HT(1B)R agonist. Interestingly, inverse H1R agonists inhibit constitutively H1R-mediated as well as 5-HT(1B)R agonist-induced signaling in cells co-expressing both receptors. This phenomenon is not solely characteristic of 5-HT(1B)R; it is also evident with muscarinic M2 and adenosine A1 receptors and is mimicked by mastoparan-7, an activator of G(i/o) proteins, or by over-expression of Gbetagamma subunits. Likewise, expression of the G(q/11)-coupled human cytomegalovirus (HCMV)-encoded chemokine receptor US28 unmasks a functional coupling of G(i/o)-coupled CCR1 receptors that is mediated via the constitutive activity of receptor US28. Consequently, constitutively active G(q/11)-coupled receptors, such as the H1R and HCMV-encoded chemokine receptor US28, constitute a regulatory switch for signal transduction by G(i/o)-coupled receptors, which may have profound implications in understanding the role of both constitutive GPCR activity and GPCR cross-talk in physiology as well as in the observed pathophysiology upon HCMV infection.     

Investigation of the role of the carboxyl-terminal tails of the alpha and beta isoforms of the human thromboxane A(2) receptor (TP) in mediating receptor:effector coupling

We have investigated the functional coupling of alpha and beta isoforms of the human thromboxane A(2) receptor (TP) to Galpha(16) and Galpha(12) members of the G(q) and G(12) families of heterotrimeric G proteins in human embryonic kidney (HEK) 293 cell lines HEK.alpha10 or HEK.beta3, stably over-expressing TPalpha and TPbeta, respectively. Moreover, using HEK.TP(Delta328) cells which over-express a variant of TP truncated at the point of divergence of TPalpha and TPbeta, we investigated the requirement of the C-tail per se in mediating G protein coupling and effector activation. Both TPalpha and TPbeta couple similarly to Galpha(16) to affect increases in inositol 1,4,5-trisphosphate (IP(3)) and mobilisation of intracellular calcium ([Ca(2+)](i)) in response to the TP agonist U46619. Whilst both TP isoforms mediated [Ca(2+)](i) mobilisation in cells co-transfected with Galpha(12), neither receptor generated corresponding increases in IP(3), indicating that the Galpha(12)-mediated increases in [Ca(2+)](i) do not involve PLC activation. Verapamil, an inhibitor of voltage dependent Ca(2+) channels, reduced [Ca(2+)](i) mobilisation in TPalpha and TPbeta cells co-transfected with Galpha(12) to approximately 40% of that mobilised in its absence, whereas [8-(N,N-diethylamino)-octyl-3,4, 5-trimethoxybenzoate, hydrochloride] (TMB-8), an antagonist of intracellular Ca(2+) release, had no effect on [Ca(2+)](i) mobilisation by either receptor isoform co-transfected with Galpha(12). Despite the lack of differential coupling specificity by TPalpha and TPbeta, TP(Delta328) signalled more efficiently in the absence of a co-transfected G protein compared to the wild type receptors but, on the other hand, displayed an impaired ability to couple to co-transfected Galpha(11), Galpha(12) or Galpha(16) subunits. In studies investigating the role of the C-tail in influencing coupling to the effector adenylyl cyclase, similar to TPalpha but not TPbeta, TP(Delta328) coupled to Galpha(s), leading to increased adenosine 3',5'-cyclic monophosphate (cAMP), rather than to Galpha(i). Whereas TP(Delta328) signalled more efficiently in the absence of co-transfected G protein compared to the wild type TPalpha, co-transfection of Galpha(s) did not augment cAMP generation by TP(Delta328). Hence, from these studies involving the wild type TPalpha, TPbeta and TP(Delta328), we conclude that the C-tail sequences of TP are not a major determinant of G protein coupling specificity to Galpha(11) and Galpha(16) members of the G(q) family or to Galpha(12); it may play a role in determining G(s) versus G(i) coupling and may act as a determinant of coupling efficiency.     

Serotonin (5-Hydroxytryptamine), a novel regulator of glucose transport in rat skeletal muscle

In this study we show that serotonin (5-hydroxytryptamine (5-HT)) causes a rapid stimulation in glucose uptake by approximately 50% in both L6 myotubes and isolated rat skeletal muscle. This activation is mediated via the 5-HT2A receptor, which is expressed in L6, rat, and human skeletal muscle. In L6 cells, expression of the 5-HT2A receptor is developmentally regulated based on the finding that receptor abundance increases by over 3-fold during differentiation from myoblasts to myotubes. Stimulation of the 5-HT2A receptor using methylserotonin (m-HT), a selective 5-HT2A agonist, increased muscle glucose uptake in a manner similar to that seen in response to 5-HT. The agonist-mediated stimulation in glucose uptake was attributable to an increase in the plasma membrane content of GLUT1, GLUT3, and GLUT4. The stimulatory effects of 5-HT and m-HT were suppressed in the presence of submicromolar concentrations of ketanserin (a selective 5-HT2A antagonist) providing further evidence that the increase in glucose uptake was specifically mediated via the 5-HT2A receptor. Treatment of L6 cells with insulin resulted in tyrosine phosphorylation of IRS1, increased cellular production of phosphatidylinositol 3,4,5-phosphate and a 41-fold activation in protein kinase B (PKB/Akt) activity. In contrast, m-HT did not modulate IRS1, phosphoinositide 3-kinase, or PKB activity. The present results indicate that rat and human skeletal muscle both express the 5-HT2A receptor and that 5-HT and specific 5-HT2A agonists can rapidly stimulate glucose uptake in skeletal muscle by a mechanism which does not depend upon components that participate in the insulin signaling pathway.     

A complex signaling cascade links the serotonin2A receptor to phospholipase A2 activation: the involvement of MAP kinases

Previous studies in our laboratory have shown that in NIH3T3-5HT2A cells, 5-HT-induced AA release is PLA2-coupled and independent of 5-HT2A receptor-mediated PLC activation. Although 5-HT2A receptor-mediated PLC activation is known to be Galphaq-coupled, much less is understood about 5-HT2A receptor-mediated PLA2 activation. Therefore, the studies presented here were aimed at elucidating the signal transduction pathway linking stimulation of the 5-HT2A receptor to PLA2 activation. By employing various selective inhibitors, toxins, and antagonistic peptide constructs, we propose that the 5-HT2A receptor can couple to PLA2 activation through two parallel signaling cascades. Initial experiments were designed to examine the role of pertussis toxin-sensitive G proteins, namely Galphai/o, as well as pertussis toxin-insensitive G proteins, namely Galpha12/13, in 5-HT-induced AA release. Furthermore, inactivation of both Gbetagamma heterodimers and Rho proteins resulted in decreased agonist-induced AA release, without having any effect on PLC-IP accumulation. We also demonstrated 5-HT2A receptor-mediated phosphorylation of ERK1,2 and p38. Moreover, pretreatment with selective ERK1,2 and p38 inhibitors resulted in decreased 5-HT-induced AA release. Taken together, these results suggest that the 5-HT2A receptor expressed in NIH3T3 cells can couple to PLA2 activation though a complex signaling mechanism involving both Galphai/o-associated Gbetagamma-mediated ERK1,2 activation and Galpha12/13-coupled, Rho-mediated p38 activation.