Agonist-like activity of antibodies directed against the second extracellular loop of the human cardiac serotonin 5-HT4(e) receptor in transfected COS-7 cells

We have previously reported that antipeptide antibodies directed against the second extracellular loop of the cardiac h5-HT4 receptor could block the activation of the L-type Ca channel in human atrial cardiomyocytes. In this paper we investigate the immunological and physiological activity of these antibodies, in a cell system expressing a larger amount of receptors than the atrial cells. The recombinant receptor was expressed at the surface of COS-7 cells under an active form (serotonin, EC50 = 1.81 x 10(-7) M), at a high level (375 +/- 25 fmol receptor/mg total protein) and was able to bind a specific ligand (GR113808) with a high affinity (Kd = 0.28 +/- 0.05 nM). In this system, the same anti-peptide antibodies used for the cardiac cells induced an "agonist-like" effect on the recombinant h5-HT4 receptor. These results are in line with those shown for others G-protein coupled receptors, as adrenoreceptors. In addition, this work showed that the effect of the antibodies is not only dependent on the epitopic region recognised but also on the molecular density and/or the cellular environment of the target receptors. Finally, our results support the hypothesis that the h5-HT4 receptor could be a new target for autoantibodies in patients with atrial arrhythmia.     

Lack of sumatriptan-induced aortic contraction or relaxation: 5-HT1B receptor protein detected in endothelium and smooth muscle of vasa vasorum but not aorta

Since 5-HT1B receptor mRNA was reported in rat aorta, and 5-HT1B receptor activation has been linked to vascular contraction, we explored sumatriptan-induced contractility and immunohistochemical density of 5-HT1B receptor protein in rat aorta. Sumatriptan (up to 10(-4) M), a 5-HT1B/1D receptor agonist, did not contract the endothelial intact or denuded rat aorta, even in the presence of L-NAME or after induction of modest tone with PGF2 alpha (10(-6) M). Sumatriptan also did not relax aortic preparations precontract with PGF2 alpha (3 x 10(-6) M) or phenylephrine (3 x 10(-7) M). Using a polyclonal antibody directed against the 5-HT1B receptor, minimal 5-HT1B receptor protein was detected in either the endothelium or smooth muscle of the rat aorta. However, dense 5-HT1B receptor protein was found in the vascular smooth muscle of the vasa vasorum supplying the aorta. Thus, the 5-HT1B receptor mRNA detected in tissue extracts of the rat aorta most likely reflects 5-HT1B receptor expression in the arterioles of the vasa vasorum. These studies support the link between the 5-HT1B receptor and vascular contraction in that the aorta with low density of 5-HT1B receptors lacked responses to sumatriptan, an agonist thought to contract blood vessels via 5-HT1B/1D receptors. Furthermore, caution must be observed when using 5-HT1B receptor mRNA to suggest receptor protein in tissues since this RT-PCR product may be derived predominantly from small blood vessels.     

Phosphorylation of the 5-hydroxytryptamine3 (5-HT3) receptor expressed in HEK293 cells

The 5-hydroxytrptamine3 (5-HT3) receptor is a pentameric complex belonging to the family of ligand-gated ion channels. A variety of studies have suggested that phosphorylation regulates the rate of desensitisation and the size of amplitude of the receptor current. In this study we have examined the phosphorylation of the myc-tagged wild-type 5-HT3A receptor subunits from guinea-pig expressed in HEK293 cells (human embryonic kidney). Stably transfected cells were metabolically labelled with 32P-phosphoric acid. The results of immunoprecipitation and autoradiography demonstrate that both splicc variants of the 5-HT3A receptor subunit are phosphorylated in HEK293 cells. Site-specific mutagenesis revealed that phosphorylation occurs at serine 409, a potential target of protein kinase A. Thus the 5-HT3 receptor might be modulated by intracellular pathways, that allow variable 5-hydroxytryptamine action as responses to different extracellular stimuli.     

Polyclonal antibody effects on the human cardiac 5-HT4(e) receptors depend upon the expression system

The initial objective of this work was to examine the effects of an antibody (Anti-G21V) directed against the second extracellular loop of human heart 5-HT4 receptors expressed in Chinese hamster ovary (CHO) cells. The antibody anti-G21V had no effect upon either basal cAMP-or 5-HT-evoked increases in cAMP in CHO cells, whereas it had shown an agonist-like effect in COS-7 cells. Analysis of agonist fractions of h5-HT4(e) receptors in CHO and COS-7 cells revealed that equilibrium constant could underlie the different responses of the receptor toward the anti-G21V antibody. Therefore, different expression systems could give rise to functional differences in 5-HT4 receptor behavior.     

Enhancement of 5-hydroxytryptamine3A receptor function by phorbol 12-myristate, 13-acetate is mediated by protein kinase C and tyrosine kinase activity

The effects of phorbol 12-myristate, 13-acetate (PMA) on 5-hydroxytryptamine (5-HT)-evoked ion currents in the mouse 5-HT3A receptor were examined. Perfusion with PMA caused a concentration dependent potentiation of 5-HT mediated currents and increased both potency and efficacy of 5-HT at the 5-HT3A receptor expressed in Xenopus oocytes. Enhancement of receptor function was partially blocked by injection of oocytes with PKCI, the peptide inhibitor of protein kinase C (PKC). Mutation of all 12 intracellular serine and threonine residues to alanine was without effect on PMA-induced potentiation of 5-HT elicited currents. Mutation of tyrosine 458 in the 5-HT3A receptor lacking intracellular serines and threonines reduced the PMA-induced potentiation of 5-HT evoked currents by approximately 55%. In contrast, mutation of tyrosine 458 in the wild-type receptor did not alter PMA-induced enhancement. The tyrosine kinase inhibitor, lavendustin A, reduced the enhancement of 5-HT3A receptor mediated currents by PMA in the mutant 5-HTA3A receptor containing no intracellular serine or threonine residues, but not in the wild-type receptor. Thus, the role of intracellular serines and threonines is redundant with that of tyrosine, suggesting that these two components act through a similar pathway in response to PMA treatment.     

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.     

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.     

Stable expression of human homomeric and heteromeric AMPA receptor subunits in HEK293 cells

Human homomeric and heteromeric alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-type glutamate receptors (GluRs) were stably expressed in HEK293 cells with cDNAs encoding the flip splice variant of GluR1, GluR2, GluR3, GluR4 subunit, and the GluR1/GluR2, GluR3/GluR2, and GluR4/GluR2 combination. The lethal combination of GluR2 and GluR4 subunits was found in high expression levels of both receptors. The AMPA-evoked current voltage relationships demonstrated the functional channel properties, such as a double rectification in GluR1, GluR3, and GluR4 receptors, and a linear relation in receptors assembled from GluR2 alone and coexpression of GluR2 with the other subunits. All the transfectants exhibited higher selectivity for AMPA than glutamate in dose-dependent current responses. [3H]AMPA binding revealed that the homomeric and heteromeric receptors displayed a single binding site in Scatchard analysis, with dissociation constant (Kd) values in the range of 14.5-49.3 nM. The Bmax values were in the range of 0.57-7.66 pmol/mg protein. The ligand displacement potency for [3H]AMPA binding was CNQX > glutamate > NS257 in all of the transfectants. These results suggest that stable transformants expressing human homomeric and heteromeric AMPA receptors will be useful tools to define selectivity and potential site of action for AMPA receptor modulators.     

Unmasking different constitutive activity of four chemoattractant receptors using Na+ as universal stabilizer of the inactive (R) state.

Neutrophils express receptors for the chemoattractants N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) complement C5a, leukotriene B4 (LTB4) and platelet-activating factor (PAF). The aim of this study was to analyze the constitutive activity of chemoattractant receptors by studying binding of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) to the G-protein Gi alpha 2 beta 1 gamma 2 expressed in Sf9 cells. We used Na+ as modulator of constitutive activity because there are no known inverse agonists for the C5a receptor (C5aR), LTB4 receptor (BLTR) and PAF receptor (PAFR). In the absence of NaCl, PAF and LTB4 exhibited larger relative stimulatory effects on GTP gamma S binding than fMLP and C5a. NaCl showed larger inhibitory effects on basal GTP gamma S binding in membranes expressing the formyl peptide receptor (FPR) and C5aR than in membranes expressing BLTR and PAFR. The order of potency of NaCl at inhibiting basal GTP gamma S binding was FPR > C5aR approximately BLTR > PAFR. As a result of the inhibitory effect of NaCl on basal GTP gamma S binding, the relative stimulatory effects of agonists were increased. By quantitatively analyzing the expression levels of chemoattractant receptors and Gi alpha 2 and the stoichiometry of receptor/G-protein coupling we obtained no evidence for structural instability of constitutively active receptors and catalytical G-protein activation. Taken together, the FPR and C5aR exhibit higher constitutive activity than the BLTR and PAFR. Na+ acts as a universal stabilizer of the inactive (R) state in chemoattractant receptors. The different potencies of NaCl at suppressing basal G-protein activity with different receptors indicate that chemoattractant receptors differ from each other in their Na(+)-affinity.     

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.     

Beta3 receptors mediate relaxation in stomach fundus whereas a fourth beta receptor mediates tachycardia in atria from transgenic beta3 receptor knockout mice

Pharmacological studies have revealed a non-beta1, beta2 or beta3 adrenergic receptor that mediates tachycardia in rat and human atria. The present studies utilized transgenic mice that lack the rodent beta3 receptor to explore, in a more definitive fashion, whether a non-beta1, beta2 or beta3 receptor can mediate atrial tachycardia. Insofar as the rat stomach fundus possesses a beta3 receptor mediating relaxation, we examined the stomach fundus from beta3 receptor knockout mice for the presence or absence of the beta3 relaxant receptor. Contractile responses to carbamylcholine were similar in potency and magnitude between mouse stomach fundus from wild type and beta3 receptor knockout animals. However, the classical beta3 receptor agonist CL316243, (10(-8)-10(-6)M) relaxed stomach fundus from wild type mice, but not from the beta3 receptor knockout animals. These data provide functional evidence for the absence of the beta3 receptor in beta3 receptor knockout animals and support the role of beta3 receptors mediating relaxation in mouse stomach fundus. Atria from mice lacking the beta3 receptor responded similarly (in potency and maximal increase in heart rate) to isoproterenol (10(-9)-10(-6)M) as atria from wild type mice. Furthermore, propranolol (3 x 10(-7) M) produced a dextral shift in the concentration response to isoproterenol in atria from both the beta3 receptor knockout and wild type mice with negative log K(B) values of 8.03 and 8.09, respectively. Thus, beta receptors mediating tachycardia to isoproterenol are intact and respond similarly in atria from both knockout and wild type mice. Furthermore, CGP12177, a prototypic 'atypical' beta receptor agonist produced tachycardia with a similar EC50 and maximal response in atria from both the wild type and beta3 receptor knockout mice. Cyanopindolol was a partial agonist relative to CGP12177 in both wild type and beta3 receptor knockout mice. Tachycardia to CGP12177 and cyanopindolol was not blocked by propranolol (3 x 10(-7) M) in atria from either group. These data provide definitive evidence that the receptor mediating tachycardia to CGP12177 and to cyanopindolol in atria from the transgenic beta3 receptor knockout mice is neither the beta1, beta2, nor beta3 adrenergic receptor.     

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.     

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.     

High efficiency activation of L-type Ca2+ current by 5-HT in human atrial myocytes

In human atrial myocytes, serotonin rather than sympathetic, stimulation is more frequently associated with atrial fibrillation. So does the arrhythmogenic effect of serotonin result from the mechanism of action of the receptor or the context of its action upon cardiac myocytes? The capacity of agonists to produce cAMP followed the sequence 5-HT < Iso < Forskolin to increase ICaL with 5-HT = Iso = Forskolin. The simultaneous application of threshold concentrations of 5-HT and Iso maximally increased ICaL. We will show that the effect of 5-HT upon human atrial myocytes is an imbalance between low production of cAMP and maximal activation of ICaL.     

The parathyroid hormone 2 (PTH2) receptor

The human PTH2 receptor binds and is activated at high potency by PTH and by the recently discovered peptide tuberoinfundibular peptide of 39 residues (TIP39). Rat and zebrafish PTH2 receptors are more weakly activated by PTH, suggesting that TIP39 may be the natural ligand for the PTH2 receptor. Unlike the PTH1 receptor, the PTH2 receptor interacts extremely weakly with parathyroid hormone-related peptide (PTHrP). The PTH2 receptor is strongly coupled to stimulation of cAMP accumulation, and more weakly, in a cell-specific manner to increases in intracellular calcium concentration. A variety of evidence supports the general model of receptor amino terminal sequences binding ligand carboxyl terminal sequences with high affinity, and ligand amino terminal sequences activating the receptor through interaction with the "juxtamembrane" portion of the receptor. The receptor is present at greatest levels in the nervous system. It is expressed in scattered cells in the cerebral cortex and basal ganglia and at relatively high abundance in the septum, midline thalamic nuclei, several hypothalamic nuclei, and the dorsal horn of the spinal cord. Peripherally, expression in pancreatic islet somatostatin cells is most dramatic. Functional hypotheses based on the receptor's distribution are being tested. Recent data support involvement in hypothalamic releasing-factor secretion and pain.     

Solubilization and purification of the human ETB endothelin receptor produced by high-level fermentation in Pichia pastoris

In the present report, the successful solubilization and purification of the ETB receptor heterologously produced in the methylotrophic yeast P. pastoris is described for the first time. In comparison to the baculovirus system where successful production, solubilization and purification have already been reported, handling and up-scaling of recombinant P. pastoris cells was much easier and less time consuming. Recombinant P. pastoris clones producing two different ETB receptor constructs were grown in a fermenter to a density of about 360 g/l. After induction with methanol, a production level of maximally 45 pmol/mg was obtained, a value which is in the range of that reported for baculovirus-infected insect cells. A method for the large-scale preparation of membranes was established. Solubilization of the recombinant ETB receptor was achieved with the detergent n-dodecyl-/beta-D-maltopyranoside. The stability of the solubilized and ligand-bound receptor was examined in detail. Subsequently, two purification methods for two different receptor constructs were tested and a large-scale procedure for isolation of recombinant receptor was established. In general, the purification methods described herein will be adaptable to other G protein-coupled receptors heterologously produced in heterologous expression systems including P. pastoris.     

Molecular modelling of the 5-hydroxytryptamine receptors.

Molecular models of the 5-hydroxytryptamine receptors are reviewed with particular emphasis upon ligand receptor interactions. The use of such models in the areas of drug design and the design and rationalisation of experiments is discussed. It is clear that many groups have modelled the 5-HT receptors using either the bacteriorhodopsin structure, rhodopsin electron density map or rhodopsin alpha-carbon template. Although it is difficult to assess the accuracy of these models it is expected that the rhodopsin based models show improved accuracy over the bacteriorhodopsin based models. Nevertheless models that are thoroughly validated with experimental mutagenesis and ligand binding data are useful in a qualitative sense providing ideas upon target compounds to synthesise and the design and rationalisation of mutagenesis experiments.     

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.