Functional expression of rat VPAC1 receptor in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae was examined as host for heterologous expression of the G protein-coupled VPAC1 receptor. Rat VPAC1 receptor cDNA and two chimeric constructs encoding the yeast mating factor pre-pro alpha-leader peptide fused in-frame to rat VPAC1 receptor were expressed in yeast cells under control of a galactose inducible promoter. The rat VPAC1 receptor was fused to the HSV tag epitope to ensure proper immunological detection of the receptor. Crucial conditions for high-level expression of active rat VPAC1 receptor included growth in amino acid supplemented minimal medium, fusion to the yeast alpha-leader peptide and a temperature shift from 30 degrees C to 15 degrees C before promoter induction. Western blotting showed that the expressed receptor was highly glycosylated and a band of 47 kDa was obtained upon endoglycosidase H treatment. Binding with radioiodinated vasoactive intestinal polypeptide revealed a KD of 2.5 nM and an IC50 of 15 nM when displacing with unlabeled vasoactive intestinal polypeptide. VPAC1 receptor density quantified by Western blotting was 510 pmol/mg membrane protein of which only 66 pmol/mg were able to bind vasoactive intestinal polypeptide.     

Receptors for calcitonin gene-related peptide,adrenomedullin, and amylin: the contributions of novel receptor-activity-modifying proteins

The discovery of receptor-activity-modifying proteins (RAMP) revealed a new principle for the function of G protein-coupled receptors. The initially orphan calcitonin receptor-like receptor (CRLR) was identified as a CGRP receptor when coexpressed with RAMP1. The same receptor is specific for adrenomedullin (ADM) in the presence of RAMP2. Calcitonin receptors (CTR) with 60% homology to the CRLR predominantly recognize calcitonin in the absence of RAMP. An amylin/CGRP receptor was recognized when a calcitonin receptor (CTR) was coexpressed with RAMP1. In the presence of RAMP3, the CTR only interacts with amylin. Noncovalent association of the RAMP with the CRLR or the CTR reveals heterodimeric RAMP/receptor complexes at the cell surface. Thus, two Class II G protein-coupled receptors, the CRLR and CTR, associate with three RAMP to form high affinity receptors for CGRP, ADM, or amylin. Here, the molecular composition and the functional properties of these receptors is reviewed.     

The bradykinin B2 receptor couples less efficiently than the angiotensin AT1 receptor to the G protein Gq in transiently transfected COS-7 cells

The purpose of this study was to compare the efficiency of two different Gq protein-coupled receptors (AT1 receptor for angiotensin II and B2 receptor for bradykinin) to activate phospholipase C (PLC). When the receptors were expressed at a similar level of 0.5 pmol/mg of protein, inositol trisphosphate (IP) accumulation elicited by AT1 receptor was four times higher than that elicited by B2 receptor. Genistein and pertussis toxin did not modify AT1 receptor- or B2 receptor-induced IP accumulation. These results indicate that in COS-7 cells, the two receptors activate PLC beta through G proteins of the Gq family. AT1 or B2 receptors were co-expressed with the alpha subunit of either Gq or G11. Both alpha subunits potentiated to the same extent AT1 receptor-induced IP accumulation. alpha 11 was also as efficient as alpha q to potentiate B2 receptor-induced response. Interestingly, however, the potentiating effect of alpha q and alpha 11 was more important (by 5-fold) on AT1 receptor-mediated response than on B2 receptor-mediated response. These results demonstrate that the extent of activation of PLC beta by different Gq-coupled receptors depends on the level of expression of these receptors and on their coupling efficiency. These are important parameters that determine the relative contribution of specific hormones to different biological processes.     

Pharmacological analysis of the contractile role of M2 and M3 muscarinic receptors in smooth muscle

Muscarinic receptors expressed on smooth muscle cells are primarily of the M(2) and M(3) subtypes. The M(3) subtype triggers contraction through an interaction with G(q) proteins to stimulate phosphoinositide hydrolysis and mobilize Ca(2+). In contrast, activation of M(2) receptors modulates contraction by preventing relaxation or by potentiating M(3) receptor-mediated contractions, which enhances heterologous desensitization. These effects can be explained by the coupling of M(2) receptors to G(i) proteins that mediate an inhibition of adenylyl cyclase and calcium-activated potassium channels. The pharmacological antagonism of a response mediated through an interaction between M(2) and M(3) receptors has been shown to resemble the profile of the directly acting receptor (M(3)), primarily, and not that of the conditional receptor (M(2)). Evidence for a contractile role of the M(2) receptor has been obtained by inactivating its signaling pathway with pertussis toxin or by measuring contractile effects of muscarinic agonists after M(3) receptors have been covalently inactivated. Under these conditions, M(2) receptors have been shown to mediate an inhibition of the relaxant effects of agents, like isoproterenol, on the contractile effects of nonmuscarinic spasmogens. Muscarinic M(2) and M(3) receptor knockout mice are useful tools for exploring interactions between these receptors in smooth muscle.     

Accessory proteins for G protein-signaling systems: activators of G protein signaling and other nonreceptor proteins influencing the activation state of G proteins

Heterotrimeric G proteins are key transducers for signal transfer from outside of the cell. In addition to their regulation by the superfamily of G protein-coupled receptors, many if not all of the subtypes of heterotrimeric G proteins are also regulated by additional accessory proteins that influence guanine nucleotide binding and/or hydrolysis or subunit interactions. Activators of G protein signaling (AGS1-3) refer to a functionally defined group of proteins that activate G protein-signaling systems in the absence of a classical G protein-coupled receptor. AGS and related proteins provide unexpected insights into the regulation of the G protein activation/deactivation cycle and the functional roles of G proteins. These proteins likely play important roles in the generation of signaling complexes, the positioning of signaling proteins within the cell, and in biological roles of G proteins unrelated to a cell surface receptor. As such, these proteins and the concepts advanced with their discovery provide unexpected avenues for therapeutics and understanding disease mechanisms.     

Regulation of receptor-coupling to (multiple) G proteins. A challenge for basic research and drug discovery

G protein-coupled receptors induce intracellular signals via interaction of with cytosolic/peripheral membrane proteins, mainly G proteins. There has been much debate about the mode of interaction between the receptors, G proteins and effectors, their mobility and the ways of determining the specificity of interaction. Additional complexity has been added to system upon the discovery of i) coupling of single receptors to several G proteins and ii) active direction of this by different ligands (stimulus trafficking). These data suggest that the most primary unit in the signal transduction is the receptor complexed with a specific G protein, making the investigation of the mechanism of receptor-G protein selection and interaction even more important. In this review, I will summarize the general knowledge of receptor interaction with G proteins and effectors and the ways of investigating this.     

Coupling of the expressed cannabinoid CB1 and CB2 receptors to phospholipase C and G protein-coupled inwardly rectifying K+ channels.

Signaling of the cannabinoid CB1 and CB2 receptors through phospholipase C (PLC) and G protein-coupled inwardly rectifying K+ channels (GIRK) was studied after their expression in COS7 cells and Xenopus oocytes. The CB1 or CB2 receptor was co-expressed with alpha subunits of the Galphaq family (Galphaq, Galpha11, Galpha14, Galpha15 and Galpha16) in COS7 cells. Receptor-dependent activation of PLC was observed after co-expressing the CB1 receptor with Galpha14, Galpha15 or Galpha16 but not with Galphaq or Galpha11. Co-expression of Gbeta1 and Ggamma2 abolished the activation, indicating that the activation was mediated by Galpha. PLC activation was not observed when the CB2 receptor was expressed alone or co-expressed with any of the above Galpha subunits. Coupling to GIRK was observed with both CB1 and CB2 receptors after expression in Xenopus oocytes. Significantly larger currents were induced when the receptor was co-expressed with both GIRK1 and GIRK4 than with either GIRK alone. Co-expression of Galpha transducin with the receptor significantly reduced the K+ currents, indicating that GIRK activation was mediated by Gbetagamma but not by Galpha. These findings suggest two new signaling pathways for the cannabinoid receptors.     

Conserved sequence motifs of olfactory receptor-like proteins may participate in upstream and downstream signal transduction.

Olfactory receptor-like proteins (ORLs) are seven transmembrane domain G protein-coupled receptors. We hypothesize that, in contrast with the hypervariable regions that may interact with a variety of odor ligands, the external and internal segments of the ORLs contain conserved regions that may interact with conserved olfactory binding proteins or direct axon guidance, and with G proteins, respectively. To test this hypothesis, a comprehensive analysis using the multiple expectation maximization for motif elicitation discovery tool was performed in all the full-length ORL clones deposited in the public section of the olfactory receptor database. Ten motifs have been identified that are present in all the olfactory receptors, in the same order, and are not present in other G protein-coupled receptors. These motifs are concentrated either in the extracellular-most or the intracellular-most parts of the receptors. The generality of these motifs was verified by their existence in the partial ORLs and 28 newly identified human receptors. The existence and localization of these motifs, suggest that they may be involved in the interactions of the receptors with their upstream and downstream signaling partners. In addition, the motifs present an additional to overall homology criterion for ORL family definition.     

Functional domains of delta- and mu-opioid receptors responsible for adenylyl cyclase inhibition

Opioid receptors (delta, mu) belong to the large superfamily of G protein coupled receptors that inhibit adenylyl cyclase. We have studied the effects of seven synthetic peptides representing selected cytoplasmic regions of the murine delta-opioid receptor on forskolin-mediated adenylyl cyclase activity in membranes of D2 and Neuro(2A) cells stably expressing the delta- and mu-opioid receptors respectively. The entire third intracellular loop (i3), its amino-terminal portion (i3.1) and the carboxyl-terminal region of the second cytoplasmic loop (i2.2) enhanced dose-dependently the agonist-mediated inhibition of cAMP accumulation. The peptide-mediated effects are blocked by pertussis toxin treatment and are not observed in parental cells that lack these receptors. The inhibitory effects of the peptides on adenylyl cyclase were markedly attenuated when membranes from D2 and Neuro(2A) cells were preincubated with antisera against Gi(2) alpha and G beta subunits of G proteins. Our results provide evidence on domains of the delta- and mu-opioid receptors responsible for adenylyl cyclase inhibition.     

Cellular signaling mechanisms for muscarinic acetylcholine receptors

Signaling pathways for muscarinic acetylcholine receptors (mAChRs) include several enzymes and ion channels. Recent studies have revealed the importance of various isoforms of both alpha and betagamma subunits of G proteins in initiation of signaling as well as the role of the small monomeric G protein, Rho, in the activation of phospholipase D. Modulation of adenylyl cyclase activity by mAChRs appears more diverse as the interaction of various receptor subtypes with the many isoforms of the enzyme are studied. Both alpha and beta subunits of G(i/o) may be involved. Some mAChR responses arise through release of nitric oxide from nitrergic nerves, including salivary gland secretion and hippocampal slow wave activity. mAChRs utilize a variety of intracellular pathways to activate various mitogen-activated protein kinases. The kinases are involved in cholinergic regulation of kidney epithelial function, catabolism of amyloid precursor protein, hippocampal long-term potentiation, activation of phospholipase A(2), and gene induction. mAChR activation can also stimulate or inhibit cellular growth and apoptosis, dependent on prior levels of cellular activity. Modulation of ion channels by mAChR agonists appears increasingly complex, based on recent studies. K(+) channels may be activated by M(2) and M(4) mAChR stimulation, although in the rat superior cervical ganglion topographical constraints appear to limit the effect to the M(2) mAChR. Another ganglionic K(+) current, the M current, is inhibited by M(1) mAChR activation, but in murine hippocampus inhibition involves another receptor subtype. R-type Ca(2+) channels are both facilitated and inhibited by M(1) and M(2) mAChRs; facilitation being more pronounced with activation of M(1) mAChRs and inhibition with M(2) mAChRs.     

Unaltered agonist potency upon inducible 5-HT7(a) but not 5-HT4(b) receptor expression indicates agonist-independent association of 5-HT7(a) receptor and Gs

We compared adenylyl cyclase (AC) activation by the G protein-coupled human serotonin (5-HT) receptors 5-HT4(b) and 5-HT7(a) using an ecdysone-inducible expression system, which allowed for reproducible expression of increasing receptor densities in clonal HEK293 (EcR293) cell lines. Low constitutive expression of receptors (2-70 fmol/mg protein) was observed and could be titrated up to 50-200-fold (approximately 400-7000 fmol/mg protein) by the ecdysone analogue ponasterone A. Although 5-HT-stimulated AC activity increased with receptor density, interclonal variation precluded comparisons of coupling efficiency. Interestingly, the potency of 5-HT to stimulate AC increased with increasing receptor density only in clones expressing 5-HT4(b) receptors. The potency for 5-HT did not change in clones expressing 5-HT7(a) receptors, even though 5-HT-stimulated AC activity approached asymptotic levels. This indicates that potency of 5-HT for stimulation of AC through the 5-HT7(a) receptor is independent of receptor-Gs stoichiometry and is consistent with a model where the 5-HT7(a) receptors are tightly associated with G protein, independent of agonist binding. This supports the existence of a complex between inactive receptor and G protein, as predicted by the cubic ternary complex model. In such a system, spare receptors do not lead to increased potency of an agonist with increased receptor density.     

Monospecific antibodies as probes for the stoichiometry of recombinant GABA(A) receptors

GABA(A) receptors composed of alpha1beta3 gamma2 and alpha1beta3 subunits were expressed in insect Sf9 cells and solubilized in 1% Triton X100. In sucrose density gradients, [3H]-Ro15-1788 binding activity, in the case of alpha1beta3 gamma2, and [3H]-muscimol binding activity, in the case of alpha1beta3 containing receptors sedimented as a single sharp peak suggesting the formation of receptors containing a defined number of subunits. When alpha1beta3gamma2 -containing receptors were incubated with an alpha-subunit specific antibody (bd24), a single class of antibody receptor complex was formed irrespective of the receptor-antibody ratio. This is consistent with two alpha subunits cross-linked within the receptor by the antibody. Similar results were obtained using a beta-subunit specific antibody (bd17). Several classes of antibody-receptor complex were formed when receptors were pre-incubated with a gamma specific antibody (anti gamma(2) 1-15 Cys). This profile is consistent with the presence of a single gamma subunit in each complex. Experiments with alpha1beta3 subunit containing receptors and antibody bd24 produced a profile similar to that seen with alpha1beta3 gamma2 receptors, consistent with two alpha subunits per receptor complex. In this case, the anti-beta subunit antibody, bd17, produced a unique and complex profile consistent with three beta subunits per receptor. This method permits the rapid determination of subunit stoichiometries of homogeneous receptor populations     

GABA(A) receptor modulation in rat cerebellum granule cells

The inhibitory GABA(A) receptor is a key element in determining the pattern of nerve cell electrical activity. Thus, modulation of its function is of paramount impact in shaping neuronal functional activity under physiological and pathological conditions. This applies to cerebellar granule neurons as to all the other neurons in the brain. The culture of cerebellar granules from newborn rats is a convenient means by which to approach these cells for electrophysiological studies provided that they maintain, as far as GABA(A) receptors are concerned, the same characteristics as in situ. Thus, the regulation of GABA(A) receptor activity in these neurons has been studied by the patch-clamp technique, both in the whole-cell and outside-out configuration. An obvious first level of control of such receptors' activity is their desensitization under continued agonist application, with biphasic kinetics. The data do not allow one to conclude whether one is dealing with two different populations of receptors or with a single population with two desensitization phases; although the presence of two GABA(A) receptor populations is suggested by a host of observations. The granule cell GABA(A) receptors are modulated by changes in extracellular pH with lower pH resulting in an enhanced receptor activity. They display, under the conditions of whole-cell recording, a run-down phenomenon which is most probably due to a tyrosine phosphatase activity which is in turn under control by a protein serine kinase. Thus, in situ tyrosine phosphorylation is a key element in determining the efficiency of GABA mediated inhibition. Activation of protein kinase A or protein kinase G (PKG) down-regulates GABA(A) receptors' activity. This last event is involved in the depression of those receptors' activity by L-arginine via the production of nitric oxide. In addition, the activity of calmodulin-activated adenylate cyclase I is controlled by GABA(B) receptors. Dendritic GABA(A) receptor activity is partially blocked by previous activation of N-methyl-D-aspartate (NMDA) receptors via calcineurin mediated dephosphorylation/activation of protein tyrosine phosphatase and concomitant production of nitric oxide and PKG activation. The site phosphorylated by PKG is evidently not available for calcineurin-mediated serine dephosphorylation, due to calcineurin-specific membrane localization in respect of the GABA(A) receptor. Overall, a complex network of biochemical signals appear to keep granule cells GABA(A) receptors under a fine balance between up- and down-regulatory mechanisms. The overall data appear also to indicate the presence of two GABA(A) receptor populations: a dendritic one which can be modulated by Ca++ entering via NMDA receptors and a cell body one. The two populations are probably different in terms of desensitization kinetics and benzodiazepine sensitivity.     

Reconstitution of human 5-hydroxytryptamine5A receptor--G protein coupling in E. coli and Sf9 cell membranes with membranes from Sf9 cells expressing mammalian G proteins.

The human 5-hydroxytryptamine5A (h5-ht5A) receptor was expressed in Escherichia coli (h5-ht5A-E. coli) to verify its pharmacological profile in the absence of G proteins. In addition, the ability of the h5-ht5A receptor to interact with mammalian Gi/o and Gs proteins was investigated by a new reconstitution approach. Agonists displayed lower affinities for h5-ht5A-E. coli than for stably transfected h5-ht5A-HEK 293 cells, due to the absence of G protein coupling in E. coli. Lysergic acid diethylamide behaved as a neutral antagonist, showing equal affinities for the G protein-coupled and the uncoupled receptor. To analyze the G protein coupling behavior of the h5-ht5A receptor, h5-ht5A-E. coli membranes or h5-ht5A-Sf9 insect cell membranes were fused by vortexing to membranes from baculovirus-infected Sf9 cells expressing mammalian G proteins. The ability of the h5-ht5A receptor to differentiate between Gi/Go/Gz and Gs proteins was explored by investigation of agonist binding affinities and agonist-induced stimulation of [35S]GTP gamma S binding. The h5-ht5A receptor failed to interact with Gz and Gs proteins and coupled equally well to Gj and Go proteins to form a complex with high affinity for agonists. Under the applied conditions, however, Gi proteins were found to be better activated than Go proteins in the [35S]GTP gamma S binding assay.     

Heterologous expression of G protein-coupled receptors in U-2 OS osteosarcoma cells

Recombinant baculoviruses, in which the insect cell-specific polyhedrin promoter has been replaced with a mammalian cell-active expression cassette (BacMam viruses), are efficient gene delivery vehicles for many mammalian cell types. BacMam viruses have been generated for expression of G protein-coupled receptors (GPCRs) and used to establish Ca2+mobilization assays in HEK-293 human embryonic kidney cells and U-2 OS human osteosarcoma cells. U-2 OS cells are highly susceptible to BacMam-based gene delivery and lack many of the endogenous receptors present on HEK-293 and other mammalian cell lines typically used for heterologous expression of GPCRs. U-2 OS cells were found to have a null background for muscarine, ADP, ATP, UTP, UDP, and lysophosphatidic acid (LPA). Consequently, U-2 OS cells transduced with BacMam constructs encoding the muscarinic acetylcholine receptors (M1, M2, M3, M4, and M5subtypes), the P2Y receptors (P2Y1, P2Y2), or the LPA receptors (EDG-2, EDG-7) were used for the establishment of whole-cell Ca2+mobilization assays, assays that cannot readily be established in HEK-293 cells. U-2 OS cells were susceptible to simultaneous expression of multiple genes delivered by BacMam vectors. In U-2 OS cells the functional expression of the Gi-coupled M2and M4receptors was dependent on co-expression of the receptor and a G protein chimera, both of which were delivered to the cells via BacMam viruses. The use of U-2 OS cells and BacMam-based gene delivery has facilitated development of whole-cell-based GPCR functional assays, especially for P2Y, muscarininc acetylcholine, and LPA receptors.     

Patterns of cyclic AMP formation by coexpressed D1 and D2L dopamine receptors in HEK 293 cells

Recent studies using immunofluorescence confocal microscopy (Aizman et al., Nature neuroscience (2000) 3, 226-230) present compelling evidence for colocalization of D1 and D2 dopamine receptors on neurons in the striatum and nucleus accumbens. To examine some of the biochemical consequences of colocalization we coexpressed the D1 and D2 dopamine receptors in HEK293 cells. Dopamine D1 and D2 receptors couple to stimulation and inhibition of adenylyl cyclase, respectively. In cells expressing only the D1 receptor, dopamine stimulated cAMP formation with an EC50 of 2.15 nM. In cells expressing only the D2L receptor, dopamine inhibited cAMP formation by 80% with an EC50 of 0.02 nM. The effect of dopamine on the D2L receptor was antagonized by the selective antagonist spiperone with an IC50 of 0.31 nM. In cells coexpressing both the D1 and D2L receptors, dopamine caused an increase in cAMP that was only 20% of that observed with the D1 receptor alone. In this case, increasing concentrations of spiperone caused a change in the dose-response curve from hyperbolic to bell-shaped as the concentration of spiperone was increased. Using pharmacological constants determined from studies on the individually expressed receptors, the curves obtained in cells co-expressing the two receptors could be modeled by kinetic expressions derived by summing the contributions from each receptor. The model leads to a re-interpretation of the pharmacology of dopaminergic ligands. Hence, one consequence of colocalization is that D2 receptor antagonists become functional agonists of cAMP formation.     

Titrating the expression of a Gi protein-coupled receptor using an ecdysone-inducible system in CHO-K1 cells.

Changes in receptor density are often associated with pathological conditions. For example, high levels of the G protein-coupled somatostatin receptor, sst2, have been detected in a number of malignant cell types, a characteristic feature that is routinely utilised as a diagnostic tool. However, how the increased receptor expression affects cellular function through alterations in G protein-coupling or changes in the intensity or duration of activated signalling pathways is poorly understood. The current report details the use of an ecdysone-inducible expression system in CHO-K1 cells, whereby the consequence of modulating the level of human sst2 receptor expression on specific transduction events can be examined. A time- and concentration-dependent induction of sst2 receptor expression was attained by exposure of cells to the ecdysteroid-inducing agent, muristerone A (MuA). Increases in sst2 receptor expression were determined by immunoassay, immunoblotting and immunocytochemical analysis. Maximal sst2 receptor expression was obtained after treatment of cells with 7 microM MuA for 24 h. Functionality of the sst2 receptor was assessed by immunoblot analysis of phosphorylated forms of MAP kinase. Following receptor activation, time-dependent increases in the level of MAP kinase phosphorylation were shown to correlate with the degree of sst2 receptor induction. Confirmation of receptor activation was determined by visualisation of ligand-induced redistribution of sst2 receptors from the plasma membrane to discrete intracellular compartments. However, in a series of further studies, both immunocytochemical and fluorescence-activated cell sorting (FACS) analyses demonstrated that over a prolonged period, stable receptor expression could not be maintained in CHO-K1 cells using this expression system. Thus, routine analysis of the sst2 receptor expressing cell population is required to derive comparable results between assays, especially when some assays provide information from the whole cell population whilst others are based at the single cell level. On the basis of these observations we conclude that, providing such quality control measurements are taken, the ecdysone inducible expression system is a useful tool to modulate functional sst2 receptor expression in an in vitro environment over short time periods.     

Stoichiometry of recombinant heteromeric glycine receptors revealed by a pore-lining region point mutation

Heteromeric glycine receptors mediate synaptic inhibition in the caudal areas of the adult mammalian central nervous system (CNS). These channels resemble other receptors in the nicotinic superfamily in that they are pentamers, but may differ in that they contain alpha and beta subunits in a 3:2 rather than a 2:3 ratio. Evidence in favor of a 3alpha:2beta stoichiometry of heteromeric glycine receptors comes from biochemical data and from the expression of chimeric subunits. We investigated this question using a potentially more direct approach and mutated the highly conserved hydrophobic residues in the middle (position 9') of the pore-lining domain. This mutation increases agonist potency in all channels in the nicotinic superfamily and its effects are in first approximation proportional to the number of mutant subunit incorporated into the receptor. We expressed in HEK 293 cells wild-type glycine alpha1beta receptors or receptors bearing the 9' mutation on either the alpha or the beta subunit, using an alpha:beta plasmid ratio of 1:40 in the transfection. This resulted in negligible levels of contamination by homomeric alpha1 receptors, as proven by low picrotoxin potency and by the extreme rarity of high conductances in single channel recording. Our data show that the effects of the 9' mutation on the receptor sensitivity to glycine were more marked when the alpha subunit bore the mutation. The magnitude of the leftward shift in the agonist dose-response curve for the two mutant combinations was in agreement with a subunit stoichiometry of 3alpha:2beta.