Identification and characterization of human taste receptor genes belonging to the TAS2R family

The sense of taste is a chemosensory system responsible for basic food appraisal. Humans distinguish between five primary tastes: bitter, sweet, sour, salty and umami. The molecular events in the perception of bitter taste are believed to start with the binding of specific water-soluble molecules to G-protein-coupled receptors encoded by the TAS2R/T2R family of taste receptor genes. TAS2R receptors are expressed at the surface of taste receptor cells and are coupled to G proteins and second messenger pathways. We have identified, cloned and characterized 11 new bitter taste receptor genes and four new pseudogenes that belong to the human TAS2R family. Their encoded proteins have between 298 and 333 amino acids and share between 23 and 86% identity with other human TAS2R proteins. Screening of a mono-chromosomal somatic cell hybrid panel to assign the identified bitter taste receptor genes to human chromosomes demonstrated that they are located in chromosomes 7 and 12. Including the 15 sequences identified, the human TAS2R family is composed of 28 full-length genes and 16 pseudogenes. Phylogenetic analyses suggest a classification of the TAS2R genes in five groups that may reflect a specialization in the detection of specific types of bitter chemicals.     

From ligand binding to gene expression: new insights into the regulation of G-protein-coupled receptors.

Transmembrane signaling systems relay information from the exterior to the interior of a cell, through a series of complex protein-protein interactions and second messenger cascades. One such system consists of the G-protein-coupled receptors, which interact with G proteins upon ligand binding, and in turn activate an effector molecule. The receptor is the first component in this signaling cascade and is subject to considerable regulation. Recent studies have shown that these regulatory events can occur at the levels of receptor protein modification and receptor gene expression. Interestingly, some of these processes appear to be mediated by the same second messenger systems that these receptors activate, which leads to various forms of positive and negative feedback regulation.     

Conserved Extracellular Cysteine Pair in the M3 Muscarinic Acetylcholine Receptor Is Essential for Proper Receptor Cell Surface Localization but Not for G Protein Coupling

Most G protein-coupled receptors contain a conserved pair of extracellular cysteine residues that are predicted to form a disulfide bond linking the first and second extracellular loops. Previous studies have shown that this disulfide bond may be critical for ligand binding, receptor activation, and/or proper receptor folding. However, the potential importance of the two conserved cysteine residues for proper receptor cell surface localization has not been investigated systematically. To address this issue, we used the rat M3 muscarinic receptor as a model system. Most studies were carried out with a modified version of this receptor subtype (lacking potential N-glycosylation sites and the central portion of the third intracellular loop) that could be readily detected via western blot analysis. Cys-->Ala mutant receptors were generated, transiently expressed in COS-7 cells, and then examined for their subcellular distribution and functional properties. ELISA and immunofluorescence studies showed that the presence of both conserved cysteine residues (corresponding to C140 and C220 in the rat M3 muscarinic receptor sequence) is required for efficient expression of the M3 muscarinic receptor on the cell surface. On the other hand, these residues were found not to be essential for protein stability (determined via immunoblotting) and receptor-mediated G protein activation (studied in second messenger assays). These results shed new light on the functional role of the two extracellular cysteine residues present in most G protein-coupled receptors.     

Pronounced conformational changes following agonist activation of the M(3) muscarinic acetylcholine receptor

The conformational changes that convert G protein-coupled receptors (GPCRs) activated by diffusible ligands from their resting into their active states are not well understood at present. To address this issue, we used the M(3) muscarinic acetylcholine receptor, a prototypical class A GPCR, as a model system, employing a recently developed disulfide cross-linking strategy that allows the formation of disulfide bonds using Cys-substituted mutant M(3) muscarinic receptors present in their native membrane environment. In the present study, we generated and analyzed 30 double Cys mutant M(3) receptors, all of which contained one Cys substitution within the C-terminal portion of transmembrane domain (TM) VII (Val-541 to Ser-546) and another one within the C-terminal segment of TM I (Val-88 to Phe-92). Following their transient expression in COS-7 cells, all mutant receptors were initially characterized in radioligand binding and second messenger assays (carbachol-induced stimulation of phosphatidylinositol hydrolysis). This analysis showed that all 30 double Cys mutant M(3) receptors were able to bind muscarinic ligands with high affinity and retained the ability to stimulate G proteins with high efficacy. In situ disulfide cross-linking experiments revealed that the muscarinic agonist, carbachol, promoted the formation of cross-links between specific Cys pairs. The observed pattern of disulfide cross-links, together with receptor modeling studies, strongly suggested that M(3) receptor activation induces a major rotational movement of the C-terminal portion of TM VII and increases the proximity of the cytoplasmic ends of TM I and VII. These findings should be of relevance for other family A GPCRs.     

A fluorescence resonance energy transfer-based sensor indicates that receptor access to a G protein is unrestricted in a living mammalian cell

Fluorescence recovery after photobleaching of muscarinic receptors and G protein subunits tagged with cyan or yellow fluorescent protein showed that receptors and G proteins were mobile and not immobilized on the cell membrane. The cyan fluorescent protein-tagged Galpha and yellow fluorescent protein-tagged Gbeta subunits were used to develop sensors that coupled selectively with the M2 and M3 muscarinic receptors. In living Chinese hamster ovary cells, imaging showed that sensors emitted a fluorescence resonance energy transfer signal that was abrogated on receptor activation. When sequentially activated with highly expressed muscarinic receptors and endogenous receptors expressed at low levels, sensor molecules were sensitive to the sequence of activation and the receptor numbers. The results distinguish between models proposing that receptor and G protein types interact freely with each other on the cell membrane or that they function as mutually exclusive multimolecular complexes by providing direct support for the former model in these cells.     

Structure/function relationship of proteins belonging to the family of receptors coupled to GTP-binding proteins.

The structural properties of a number of proteins belonging to the family of receptors coupled to GTP binding proteins are discussed in relation to their function. The structure of the ligand binding site and of the regions involved in coupling to the G proteins are analyzed mainly for the adrenergic and muscarinic cholinergic receptors, for which site-directed mutagenesis and chimaeric constructions have been studied. The structure of the genes are compared and the presence of various regulatory elements is discussed in relation to control of expression. Mechanism of desensitization and internalization, while mostly studied for the beta 2-adrenergic receptor, are proposed to be generally applicable to all G-protein-coupled receptors.     

Structure-function analysis of muscarinic receptors and their associated G proteins

Each member of the muscarinic receptor family (M1-M5) can interact only with a limited subset of the many structurally closely related heterotrimeric G proteins expressed within a cell. To understand how this selectivity is achieved at a molecular level, we have used the G(i/0)-coupled M2 and the Gq/11-coupled M3 muscarinic receptors as model systems. We developed a genetic strategy involving the coexpression of wild type or mutant muscarinic receptors with hybrid or mutant G protein alpha subunits to identify specific, functionally relevant receptor/G protein contact sites. This approach led to the identification of N- and C-terminal amino acids on alpha(q) and alpha(i) that are critical for maintaining proper receptor/G protein coupling. Moreover, several receptor sites were identified that are likely to be contacted by these functionally critical G alpha residues. To gain deeper insight into muscarinic receptor structure, we recently developed a cysteine disulfide cross-linking strategy, using the M3 muscarinic receptor as a model system. Among other structural modifications, this approach involves the removal of most native cysteine residues by site-directed mutagenesis, the insertion of three factor Xa cleavage sites into the third intracellular loop, and systematic 'reintroduction' of pairs of cysteine residues. Following treatment of receptor-containing membrane preparations with factor Xa and oxidizing agents, disulfide cross-linked products can be identified by immunoprecipitation and immunoblotting studies. This approach should greatly advance our knowledge of the molecular architecture of muscarinic and other G protein-coupled receptors.     

A G protein-gated K channel is activated via beta 2-adrenergic receptors and G beta gamma subunits in Xenopus oocytes

In many tissues, inwardly rectifying K channels are coupled to seven- helix receptors via the Gi/Go family of heterotrimeric G proteins. This activation proceeds at least partially via G beta gamma subunits. These experiments test the hypothesis that G beta gamma subunits activate the channel even if released from other classes of heterotrimeric G proteins. The G protein-gated K channel from rat atrium, KGA/GIRK1, was expressed in Xenopus oocytes with various receptors and G proteins. The beta 2-adrenergic receptor (beta 2AR), a Gs-linked receptor, activated large KGA currents when the alpha subunit, G alpha s, was also overexpressed. Although G alpha s augmented the coupling between beta 2AR and KGA, G alpha s also inhibited the basal, agonist-independent activity of KGA. KGA currents stimulated via beta 2AR activated, deactivated, and desensitized more slowly than currents stimulated via Gi/Go-linked receptors. There was partial occlusion between currents stimulated via beta 2AR and the m2 muscarinic receptor (a Gi/Go-linked receptor), indicating some convergence in the mechanism of activation by these two receptors. Although stimulation of beta 2AR also activates adenylyl cyclase and protein kinase A, activation of KGA via beta 2AR is not mediated by this second messenger pathway, because direct elevation of intracellular cAMP levels had no effect on KGA currents. Experiments with other coexpressed G protein alpha and beta gamma subunits showed that (a) a constitutively active G alpha s mutant did not suppress basal KGA currents and was only partially as effective as wild type G alpha s in coupling beta 2AR to KGA, and (b) beta gamma subunits increased basal KGA currents. These results reinforce present concepts that beta gamma subunits activate KGA, and also suggest that beta gamma subunits may provide a link between KGA and receptors not previously known to couple to inward rectifiers.     

Molecular mechanisms of regulation of neuronal muscarinic receptor sensitivity

Like other neurotransmitter receptors, muscarinic acetylcholine receptors are subject to regulation by the state of receptor activation. Prolonged increases in the concentration of muscarinic agonists result in a decrease in receptor density and loss of receptor sensitivity, both in vivo and in vitro. On the other hand, when the receptor is deprived of acetylcholine for a long duration in vivo, the receptor becomes more sensitive in responding to muscarinic agonists. However, it has been more difficult to demonstrate increases in receptor concentration that accompany this supersensitive state. The purpose of this review is to provide current information related to the characteristics of muscarinic receptor regulation and the molecular mechanisms underlying this phenomenon, regarding both the density of receptors and their transduction mechanisms. Furthermore, possible feedback regulatory roles of different second messenger signals are discussed. Particular emphasis is dedicated to molecular mechanisms of regulation of neuronal muscarinic receptors.     

神经活性甾体对神经元的作用

神经活性甾体是指神经组织中具有活性的甾体激素,根据甾体激素的作用机制可分为三类：（1）通过细胞表面离子通道型受体介导产生效应,这些受体包括GABAA受体,NMDA受体等。（2）通过G蛋白偶联的膜受体指导第二信使反应,再通过DNA结合蛋白,调节基因表达产生效应,（3）通过细胞内受体介导调控基因的表达产生效应,甾体激素的这些效应尤其是对离子通道型受体和G蛋白偶联型受体的调节作用,已引起重视。     

Endocytosis and recycling of muscarinic receptors

Agonist stimulation causes the endocytosis of many G protein-coupled receptors, including muscarinic acetylcholine receptors. In this study we have investigated the agonist-triggered trafficking of the M3 muscarinic receptor expressed in SH-SY5Y human neuroblastoma cells. We have compared the ability of a series of agonists to generate the second messenger Ins(1,4,5)P3 with their ability to stimulate receptor endocytosis. We show that there is a good correlation between the intrinsic activity of the agonists and their ability to increase the rate constant for receptor endocytosis. Furthermore, on the basis of our results, we predict that even very weak partial agonists should under some circumstances be able to cause substantial receptor internalization. Receptor endocytosis occurs too slowly to account for the rapid desensitization of the Ca2+ response to carbachol. Instead, receptor endocytosis and recycling appear to play an important role in resensitization. After an initial agonist challenge, the response to carbachol is fully recovered when only about half of the receptors have been recycled to the cell surface, suggesting that there is a receptor reserve of about 50%. Removal of this reserve by receptor alkylation significantly reduces the extent of resensitization. Resensitization is also reduced by inhibitors of either endocytosis alone (concanavalin A) or of endocytosis and recycling (nigericin). Finally, the protein phosphatase inhibitor calyculin A also reduces resensitization, possibly by blocking the dephosphorylation of the receptors in an endosomal compartment.     

G protein antagonists. A novel hydrophobic peptide competes with receptor for G protein binding.

A substance P (SP) analog, [D-Pro4,D-Trp7,9,10] SP4-11, is known to inhibit the actions of various structurally unrelated messenger molecules as well as SP. Our studies on the effects of this peptide on the regulation of purified G proteins by receptor showed that at least some of the biological effects of the peptide can be explained by the ability of the peptide to block the activation of G proteins by receptors. Here we report that a novel truncated SP-related peptide, pGlu-Gln-D-Trp-Phe-D-Trp-D-Trp-Met-NH2, inhibited the activation of G(i) or G(o) by M2 muscarinic cholinergic receptor (M2 mAChR) or of Gs by beta-adrenergic receptor in the reconstituted phospholipid vesicles, assayed by receptor-promoted GTP hydrolysis. The inhibition by the peptide was apparently reversible and competitive with respect to receptor binding to G proteins; the inhibition could be overcome by increasing the concentration of receptor in the vesicles and was not altered by changes in the concentration of G protein. The competing effects of the peptide were used to analyze the effect of agonist on receptor-G protein interaction. The concentration change of muscarinic agonist did not alter the inhibitory effects of the peptide on M2 mAChR-promoted GTPase by G(o), which is consistent with the idea that agonist increases the regulatory efficiency of the receptor but does not alter its affinity for G proteins. This new group of compounds (G protein antagonists) is a promising tool to study receptor-G protein interaction quantitatively.     

Excitatory amino acids: The involvement of second messengers in the signal transduction process

1. Excitatory amino acids (EAA) can activate second messenger systems in addition to a direct gating of ion channels. A discrete coupling between novel EAA receptor subtypes and second messenger systems has been previously proposed. 2. EAAs have been suggested to activate both adenylate and guanylate cyclases and also to induce phosphoinositide (PI) turnover. The increased PI turnover was observed in both central neurons and glia, and a "quisqualate-type" receptor has been most frequently involved, which may differ from the quisqualate receptor previously defined by electrophysiological studies. 3. The roles of EAA-induced calcium influx into neurons and raised intracellular calcium levels are discussed regarding the activation of phosphoinositide turnover. 4. This review examines the data supporting a link between EAA receptors and second messengers and considers whether there is any need for adopting new EAA receptor subtypes. Also, the use of the Xenopus laevis oocyte for expressing EAA receptors and studying any putative links to second messenger systems is discussed.     

Opioid receptor-coupled second messenger systems

Although pharmacological data provide strong evidence for different types of opioid receptors (e.g., mu, delta, and kappa), they share many common properties in their ability to couple to second messenger systems. All opioid receptor types are coupled to G-proteins, since agonist binding is diminished by guanine nucleotides and agonist-stimulated GTPase activity has been identified in several preparations. Moreover, all three types inhibit adenylyl cyclase. This second messenger system has been identified for opioid receptors in both isolated brain membranes and in transformed cell culture. Studies with chronic treatment with opioid agonists suggest that the coupling of receptors with G-proteins and second messenger effectors may play important roles in development of opioid tolerance.     

Acetylcholine muscarinic receptor regulation of the Ras/Raf/MAP kinase pathway

Acetylcholine muscarinic m1 receptors and m2 receptors are predominantly coupled to the heterotrimeric G proteins Gq, 11 and Gi, respectively. Stimulation of the m1 and m2 receptors in different cell types activate the Ras/Raf/MAP kinase pathway. The ability of the m1 receptor to activate the MAP kinase pathway is dependent on the isoforms of adenylyl cyclase expressed in specific cell types. Specific adenylyl cyclases respond to different signals, including calcium and protein kinase C, with increased cAMP synthesis resulting in protein kinase A activation. Stimulation of protein kinase A inhibits Raf and subsequent MAP kinase activation by G protein-coupled receptors and growth factor receptor tyrosine kinases. G protein-coupled receptors can positively and negatively regulate the responsiveness of tyrosine kinase-stimulated response pathways.     

Peptides as probes for G protein signal transduction

Triggered by agonist binding to cell surface receptors, the heterotrimeric G proteins dissociate into and βγ subunits, each activating distinct second messenger pathways. Peptides from the primary sequences of receptors, G proteins, and effectors have been used to study the molecular interactions between these proteins. Receptor-derived peptides from the second, third and fourth intracellular loops and certain naturally occurring peptides antagonize G protein interactions and can directly activate G protein. These peptides bind to G protein sites that include the N and C terminal regions of the subunit and a yet to be identified region of the β subunit. Peptides have also been useful in characterizing G protein-effector interactions. The identification of the contact sites between proteins involved in G protein signal transduction should aid in the development of non-peptide mimetic therapeutics which could specifically modify G protein-mediated cellular responses.     

Genetic evidence for involvement of type 1, type 2 and type 3 inositol 1,4,5-trisphosphate receptors in signal transduction through the B-cell antigen receptor.

Stimulation of B-cell antigen receptor (BCR) induces a rapid increase in cytoplasmic free calcium due to its release from intracellular stores and influx from the extracellular environment. Inositol 1,4,5-trisphosphate receptors (IP3Rs) are ligand-gated channels that release intracellular calcium stores in response to the second messenger, inositol 1,4,5-trisphosphate. Most hematopoietic cells, including B cells, express at least two of the three different types of IP3R. We demonstrate here that B cells in which a single type of IP3R has been deleted still mobilize calcium in response to BCR stimulation, whereas this calcium mobilization is abrogated in B cells lacking all three types of IP3R. Calcium mobilization by a transfected G protein-coupled receptor (muscarinic M1 receptor) was also abolished in only triple-deficient cells. Capacitative Ca2+ entry, stimulated by thapsigargin, remains unaffected by loss of all three types of IP3R. These data establish that IP3Rs are essential and functionally redundant mediators for both BCR- and muscarinic receptor-induced calcium mobilization, but not for thapsigargin-induced Ca2+ influx. We further show that the BCR-induced apoptosis is significantly inhibited by loss of all three types of IP3R, suggesting an important role for Ca2+ in the process of apoptosis.     

Structure-function analysis of muscarinic acetylcholine receptors

The structural basis underlying the G protein coupling selectivity of different muscarinic receptor subtypes was analyzed by using a combined molecular genetic/biochemical approach. These studies led to the identification of key residues on the receptors as well as the associated G proteins that are critically involved in determining proper receptor/G protein recognition. Mutational analysis of the m3 muscarinic receptor showed that most native cysteine residues are not required for productive receptor/G protein coupling. The putative extracellular disulfide bond was found to be essential for efficient trafficking of the receptor protein to the cell surface but not for receptor-mediated G protein activation.     

G proteins control diverse pathways of transmembrane signaling

Hormones, neurotransmitters, and autacoids interact with specific receptors and thereby trigger a series of molecular events that ultimately produce their biological effects. These receptors, localized in the plasma membrane, carry binding sites for ligands as diverse as peptides (e.g., glucagon, neuropeptides), lipids (e.g., prostaglandins), nucleosides and nucleotides (e.g., adenosine), and amines (e.g., catecholamines, serotonin). These receptors do not interest directly with their respective downstream effector (i.e., an ion channel and/or an enzyme that synthesizes a second messenger); rather, they control one or several target systems via the activation of an intermediary guanine nucleotide-binding regulatory protein or G protein. G proteins serve as signal transducers, linking extracellularly oriented receptors to membrane-bound effectors. Traffic in these pathways is regulated by a GTP (on)-GDP (off) switch, which is regulated by the receptor. The combination of classical biochemistry and recombinant DNA technology has resulted in the discovery of many members of the G protein family. These approaches, complemented in particular by electrophysiological experiments, have also identified several effectors that are regulated by G proteins. We can safely assume that current lists of G proteins and the functions that they control are incomplete.