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ABSTRACT: Small molecule fluorochromes (synonyms: biosensors, chemosensors, fluorescent probes, vital stains) are widely used to investigate the structure, composition, physicochemical properties and biological functions of living cells, tissues and organisms. Selective entry and accumulation within particular cells and cellular structures are key processes for achieving these diverse objectives. Despite the complexities, probes routinely are applied using standard protocols, often without experimenter awareness of what factors that control accumulation and localization. The mechanisms of many such selective accumulations, however, now are known. Moreover, the influence of physicochemical properties of probes on their uptake and localization often can be defined numerically, hence predicted, using quantitative structure activity relations (QSAR) models with its required numerical structure parameters (or "descriptors"). The state of the art of this approach is described. Available QSAR models are summarized for uptake into cells and localization in the cytosol, endoplasmic reticulum, generic biomembranes, Golgi apparatus, lipid droplets, lysosomes/endosomes, mitochondria, eukaryotic nuclei (histones and DNA), plasma membrane, and ribosomal RNA (cytoplasmic and nucleolar). Integration of such core models to both aid understanding and troubleshooting of current fluorescent probes and to assist the design of novel probes is outlined and illustrated using case examples. Limitations and generic problems arising with this approach and comments on application of such approaches to xenobiotics other than probes, e.g., drugs and herbicides, together with a brief note about an alternative approach to prediction, are given.
Biotechnic & Histochemistry 06/2013; · 0.67 Impact Factor
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ABSTRACT: A wide range of intracellular proteins have been demonstrated to interact with individual G protein-coupled receptors (GPCRs) and, in certain cases, to modulate their function or trafficking. However, in only a few cases have the GPCR selectivity of such interactions been investigated. Interactions between the intracellular C-terminal tails of 44 GPCRs and both neurochondrin and periplakin were assessed in pull-down studies. 23 of these interacted with neurochondrin and periplakin, 10 interacted with neither whilst nine interacted with only neurochondrin and two with only periplakin. When appropriate GIP-interacting G(q)/G(11)-coupled GPCRs were expressed in cells inducibly expressing neurochondrin or periplakin this resulted in a reduction in the increase in intracellular [Ca(2+)] in response to agonist. However, induction of neurochondrin or periplakin was without functional consequences for GPCRs with which they did not interact. Unlike intracellular [Ca(2+)] signals, induction of expression of either interacting protein did not inhibit agonist-mediated ERK1/2 MAPK phosphorylation. These data indicate that both periplakin and neurochondrin can interact with a wide range of GPCRs and modulate function selectively. Details of the structure of the intracellular C-terminal tail of individual receptors will be required to fully understand the basis of such selectivity.
Journal of Neurochemistry 02/2009; 109(1):182-92. · 4.06 Impact Factor
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G Milligan
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ABSTRACT: G protein-coupled receptors are one of the most actively studied families of proteins. However, despite the ubiquity of protein dimerization and oligomerization as a structural and functional motif in biology, until the last decade they were generally considered as monomeric, non-interacting polypeptides. For the metabotropic glutamate-like group of G protein-coupled receptors, it is now firmly established that they exist and function as dimers or, potentially, even within higher-order structures. Despite some evidence continuing to support the view that rhodopsin-like G protein-coupled receptors are predominantly monomers, many recent studies are consistent with the dimerization/oligomerization of such receptors. Key roles suggested for dimerization of G protein-coupled receptors include control of protein maturation and cell surface delivery and providing the correct framework for interactions with both hetero-trimeric G proteins and arrestins to allow signal generation and its termination. As G protein-coupled receptors are the most targeted group of proteins for the development of therapeutic small molecule medicines, recent indications that hetero-dimerization between co-expressed G protein-coupled receptors may be a common process offers the potential for the development of more selective and tissue restricted medicines. However, many of the key experiments have, so far, been limited to model cell systems. Priorities for the future include the generation of tools and reagents able to identify unequivocally potential G protein-coupled receptor hetero-dimers in native tissues and detailed analyses of the influence of hetero-dimerization on receptor function and pharmacology.
British Journal of Pharmacology 04/2008; 153 Suppl 1:S216-29. · 4.41 Impact Factor
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ABSTRACT: A wide range of techniques have been employed to examine the quaternary structure of G-protein-coupled receptors (GPCRs). Although it is well established that homo-dimerisation is common, recent studies have sought to explore the physical basis of these interactions and the role of dimerisation in signal transduction. Growing evidence hints at the existence of higher-order organisation of individual GPCRs and the potential for hetero-dimerisation between pairs of co-expressed GPCRs. Here we consider how both homo-dimerisation/oligomerisation and hetero-dimerisation can regulate signal transduction through GPCRs and the potential consequences of this for function of therapeutic medicines that target GPCRs. Hetero-dimerisation is not the sole means by which co-expressed GPCRs may regulate the function of one another. Heterologous desensitisation may be at least as important and we also consider if this can be the basis for physiological antagonism between pairs of co-expressed GPCRs. Although there may be exceptions (Meyer et al. 2006), a great deal of recent evidence has indicated that most G-protein-coupled receptors (GPCRs) do not exist as monomers but rather as dimers or, potentially, within higher-order oligomers (Milligan 2004b; Park et al. 2004). Support for such models has been provided by a range of studies employing different approaches, including co-immunoprecipitation of differentially epitope-tagged but co-expressed forms of the same GPCR, co-operativity in ligand binding and a variety of resonance energy transfer techniques (Milligan and Bouvier 2005). Only for the photon receptor rhodopsin has the organisational structure of a GPCR been studied in situ. The application of atomic force microscopy to murine rod outer segment discs indicated that rhodopsin is organised in a series of parallel arrays of dimers (Liang et al. 2003) and based on this, molecular models were constructed to try to define and interpret regions of contact between the monomers (Fotiadis et al. 2004). Only for relatively few other GPCRs are details of the molecular basis of dimerisation available but within this limited data set, recent studies on the dopamine D2 receptor suggest a means by which information on the binding of an agonist can be transmitted between the two elements of the dimer via the dimer interface (Guo et al. 2005). Although the availability of cDNAs encoding molecularly defined GPCRs has allowed high-throughput screening for ligands that modulate GPCR function, this is performed almost exclusively in heterologous cell lines transfected to express only the specific GPCR of interest. Given that the human genome contains some 400-450 genes encoding non-chemosensory GPCRs, it is clear that any individual cell of the body may express a considerable number of GPCRs. Interactions between these, either via hetero-dimerisation, via heterologous desensitisation or via the integration of downstream signals can potentially alter the pharmacology, sensitivity and function of receptor agonists and hence produce varied responses. In this article, we will use specific examples to consider the role of homo-dimerisation/oligomerisation in GPCR function and whether either direct hetero-dimerisation or heterologous desensitisation between pairs of co-expressed GPCRs affects the function of the receptor pairs.
Ernst Schering Foundation symposium proceedings. 02/2006;
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ABSTRACT: Three distinct genes encode alpha(1)-adrenoceptors. Although homodimers of each subtype have been reported, certain but not all combinations of heterodimers of the alpha(1)-adrenoceptors appear to form. Key studies in this field are reviewed and the approaches that have been applied to monitoring the selectivity and the basis of alpha(1)-adrenoceptor dimerization are discussed.
Biochemical Society Transactions 12/2004; 32(Pt 5):847-50. · 3.71 Impact Factor
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ABSTRACT: A substantial number of G-protein-coupled receptor-interacting proteins have been identified initially by the use of yeast two-hybrid screens. Using the C-terminal tail of both opioid receptors and the melanin concentrating hormone receptor-1 as bait, the actin and intermediate filament-binding protein periplakin was isolated. In each case, the site of interaction is within helix VIII of the receptor and periplakin limits agonist-mediated G-protein activation potentially by competing with G-protein for this region of the receptor.
Biochemical Society Transactions 12/2004; 32(Pt 5):878-80. · 3.71 Impact Factor
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ABSTRACT: A wide range of peptides and polypeptides can be appended to either the N- or C-terminus of G protein-coupled receptors without disrupting substantially ligand binding and signal transduction. Following fusion of fluorescent proteins, reporter gene constructs or G protein alpha subunits to the C-terminal tail of a receptor high content and G protein activation assays can be employed to identify agonist ligands. Further modification of the receptor fusions to introduce enhanced levels of constitutive activity and to physically destabilise the protein allows antagonist/inverse agonists screens to be developed in parallel. Equivalent C-terminal addition of pairs of complementary, non-functional, polypeptide fragments allows the application of enzyme complementation techniques. Introduction of N-terminal tags to receptors has also allowed the introduction of novel assay techniques based on a pH-sensitive cyanine dye. These have the capacity to overcome certain limitations of GPCR-fluorescent protein fusions.
Current Pharmaceutical Design 02/2004; 10(17):1989-2001. · 3.87 Impact Factor
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ABSTRACT: Transfection of either the alpha(1b)-adrenoreceptor or Galpha(11) into a fibroblast cell line derived from a Galpha(q)/Galpha(11) double knockout mouse failed to produce elevation of intracellular [Ca(2+)] upon the addition of agonist. Co-expression of these two polypeptides, however, produced a significant stimulation. Co-transfection of the alpha(1b)-adrenoreceptor with the palmitoylation-resistant C9S,C10S Galpha(11) also failed to produce a signal, and much reduced and kinetically delayed signals were obtained using either C9S Galpha(11) or C10S Galpha(11). Expression of a fusion protein between the alpha(1b)-adrenoreceptor and Galpha(11) allowed [Ca(2+)](i) elevation, and this was also true for a fusion protein between the alpha(1b)-adrenoreceptor and C9S,C10S Galpha(11), since this strategy ensures proximity of the two polypeptides at the cell membrane. For both fusion proteins, co-expression of transducin alpha, as a beta.gamma-sequestering agent, fully attenuated the Ca(2+) signal. Both of these fusion proteins and one in which an acylation-resistant form of the receptor was linked to wild type Galpha(11) were also targets for agonist-regulated [(3)H]palmitoylation and bound [(35)S]guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) in an agonist concentration-dependent manner. The potency of agonist to stimulate [(35)S]GTPgammaS binding was unaffected by the palmitoylation potential of either receptor or G protein. These studies provide clear evidence for coordinated, agonist-mediated regulation of the post-translational acylation of both a receptor and partner G protein and demonstrate the capacity of such fusions to bind and then release beta.gamma complex upon agonist stimulation whether or not the G protein can be palmitoylated. They also demonstrate that Ca(2+) signaling in EF88 cells by such fusion proteins is mediated via release of the G protein beta.gamma complex.
Journal of Biological Chemistry 10/2001; 276(38):35883-90. · 4.77 Impact Factor
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ABSTRACT: Fusion proteins were constructed between the delta opioid receptor and forms of the alpha subunit of G(i1) in which cysteine(351) was mutated to a range of amino acids. GDP reduced the binding of the agonist [(3)H]DADLE but not the antagonist [(3)H]naltrindole to both the receptor alone and all the delta opioid receptor-Cys(351)XaaG(i1)alpha fusion proteins. For the fusion proteins the pEC(50) for GDP was strongly correlated with the n-octanol/H(2)O partition co-efficient of G protein residue(351). Fusion proteins in which this residue was either isoleucine or glycine had similar observed binding kinetics for [(3)H]DADLE. However, the rate of dissociation of [(3)H]DADLE was substantially greater for the glycine-containing fusion protein than that containing isoleucine, indicating that more hydrophobic residues imbued greater stability to the agonist-receptor-G protein ternary complex. This resulted in a higher affinity of binding of [(3)H]DADLE to the fusion protein containing isoleucine(351). In expectation with the binding data, maximal DADLE-stimulated GTP hydrolysis by the isoleucine(351)-containing fusion protein was two-fold greater and the potency of DADLE seven-fold higher than for the version containing glycine. These results demonstrate that the stability of the ternary complex between delta opioid receptor, G(i1)alpha and an agonist (but not antagonist) ligand is dependent upon the nature of residue(351) of the G protein and that this determines the effectiveness of information flow from the receptor to the G protein.
Neuropharmacology 10/2001; 41(3):321-30. · 4.81 Impact Factor
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ABSTRACT: Recombinant RGS1, RGS16 and RGS-GAIP, but not RGS2, were able to substantially further stimulate the maximal GTPase activity of G(o1)alpha promoted by agonists at the alpha2A-adrenoreceptor in a concentration-dependent manner. Kinetic analysis of the regulation of an alpha2A-adrenoreceptor-G(o1)alpha fusion protein by all three RGS proteins revealed that they had similar affinities for the receptor-G protein fusion. However, their maximal effects on GTP hydrolysis varied over threefold with RGS16 > RGS1 > RGS-GAIP. Both RGS1 and RGS16 reduced the potency of the alpha2A-adrenoreceptor agonist adrenaline by some 10-fold. A lower potency shift was observed for the partial agonist UK14304 and the effect was absent for the weak partial agonist oxymetazoline. Each of these RGS proteins altered the intrinsic activity of both UK14304 and oxymetazoline relative to adrenaline. Such results require the RGS interaction with G(o1)alpha to alter the conformation of the alpha2A-adrenoreceptor and are thus consistent with models invoking direct interactions between RGS proteins and receptors. These studies demonstrate that RGS1, RGS16 and RGS-GAIP show a high degree of selectivity to regulate alpha2A-adrenoreceptor-activated G(o1)alpha rather than G(i1)alpha, G(i2)alpha or G(i3)alpha and different capacities to inactivate this G protein.
Journal of Neurochemistry 08/2001; 78(4):797-806. · 4.06 Impact Factor
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ABSTRACT: Although many G-protein-coupled neurotransmitter receptors are potentially capable of modulating both voltage-dependent Ca(2+) channels (I(Ca)) and G-protein-gated K(+) channels (I(GIRK)), there is a substantial degree of selectivity in the coupling to one or other of these channels in neurons. Thus, in rat superior cervical ganglion (SCG) neurons, M(2) muscarinic acetylcholine receptors (mAChRs) selectively activate I(GIRK) whereas M(4) mAChRs selectively inhibit I(Ca). One source of selectivity might be that the two receptors couple preferentially to different G-proteins. Using antisense depletion methods, we found that M(2) mAChR-induced activation of I(GIRK) is mediated by G(i) whereas M(4) mAChR-induced inhibition of I(Ca) is mediated by G(oA). Experiments with the beta gamma-sequestering peptides alpha-transducin and beta ARK1(C-ter) indicate that, although both effects are mediated by G-protein beta gamma subunits, the endogenous subunits involved in I(GIRK) inhibition differ from those involved in I(Ca) inhibition. However, this pathway divergence does not result from any fundamental selectivity in receptor-G-protein-channel coupling because both I(GIRK) and I(Ca) modulation can be rescued by heterologously expressed G(i) or G(o) proteins after the endogenously coupled alpha-subunits have been inactivated with Pertussis toxin (PTX). We suggest instead that the divergence in the pathways activated by the endogenous mAChRs results from a differential topographical arrangement of receptor, G-protein and ion channel.
European Journal of Neuroscience 08/2001; 14(2):283-92. · 3.63 Impact Factor
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ABSTRACT: Oligomerization of the human delta-opioid receptor and its regulation by ligand occupancy were explored following expression in HEK293 cells using each of co-immunoprecipitation of differentially epitope-tagged forms of the receptor, bioluminescence resonance energy transfer and time-resolved fluorescence resonance energy transfer. All of the approaches identified constitutively formed receptor oligomers, and the time-resolved fluorescence studies confirmed the presence of such homo-oligomers at the cell surface. Neither the agonist ligand [d-Ala(2),d-Leu(5)]enkephalin nor the inverse agonist ligand ICI174864 were able to modulate the oligomerization status of this receptor. Interactions between co-expressed delta-opioid receptors and beta(2)-adrenoreceptors were observed in co-immunoprecipitation studies. Such hetero-oligomers could also be detected using bioluminescence resonance energy transfer although the signal obtained was substantially smaller than for homo-oligomers of either receptor type. Signal corresponding to the delta-opioid receptor-beta(2)-adrenoreceptor hetero-oligomer was increased in the presence of agonist for either receptor. However, substantial levels of this hetero-oligomer were not detected at the cell surface using time-resolved fluorescence resonance energy transfer. These studies demonstrate that, following transient transfection of HEK293 cells, constitutively formed oligomers of the human delta-opioid receptor can be detected by a variety of approaches. However, these are not regulated by ligand occupancy. They also indicate that time-resolved fluorescence resonance energy transfer represents a means to detect such oligomers at the cell surface in populations of intact cells.
Journal of Biological Chemistry 05/2001; 276(17):14092-9. · 4.77 Impact Factor
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ABSTRACT: beta-Arrestin 1-GFP or beta-arrestin 2-GFP were coexpressed transiently with G protein-coupled receptor kinase 2 within cells stably expressing the orexin-1, apelin or melanin-concentrating hormone (MCH), receptors. In response to agonist ligands both the orexin-1 and apelin receptors were able to rapidly translocate both beta-arrestin 1-GFP and beta-arrestin 2-GFP from cytoplasm to the plasma membrane. For the MCH receptor this was only observed for beta-arrestin 2-GFP. beta-Arrestin 1-GFP translocated by the apelin receptor remained at the plasma membrane during prolonged exposure to ligand even though the receptor became internalized. By contrast, for the orexin-1 receptor, internalization of beta-arrestin 1-GFP within punctate vesicles could be observed for over 60 min in the continued presence of agonist. Co-internalization of the orexin-1 receptor was observed by monitoring the binding and trafficking of TAMRA-(5- and 6-carboxytetramethylrhodamine) labelled orexin-A. Subsequent addition of an orexin-1 receptor antagonist resulted in cessation of incorporation of beta-arrestin 1-GFP into vesicles at the plasma membrane and a gradual clearance of beta-arrestin 1-GFP from intracellular vesicles. For the melanin-concentrating hormone receptor the bulk of translocated beta-arrestin 2-GFP was maintained at concentrated foci close to, or at, the plasma membrane. These results demonstrate very distinct features of beta-arrestin-GFP interactions and trafficking for three G protein-coupled receptors for which the natural ligands have only recently been identified and which were thus previously considered as orphan receptors.
Journal of Neurochemistry 05/2001; 77(2):476-85. · 4.06 Impact Factor
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ABSTRACT: S 14506 is chemically related to the inverse agonist at 5-HT(1A) receptors, spiperone, but S 14506 behaves as one of the most potent agonists known at these receptors, both in vitro and in vivo. In hippocampal membranes, the specific binding of [(3)H]-S 14506 (K(d)=0.79+/-0.2 nM; B(max)=400+/-32 fmol/mg protein) to 5-HT(1A) receptors resembled that of an antagonist in that it was increased by GppNHp, whereas GppNHp reduced the binding of the classic agonist [(3)H]-8-OH-DPAT (K(d)=1.5+/-0.5 nM; B(max)=303+/-20 fmol/mg protein). Manganese, magnesium and calcium reduced the binding of [(3)H]-S 14506 to 5-HT(1A) receptors whereas the binding of [(3)H]-8-OH-DPAT was increased. Further, sodium markedly reduced the binding of [(3)H]-8-OH-DPAT, without affecting the binding of [(3)H]-S 14506. [(3)H]-S 14506 also bound with high affinity to h 5-HT(1A) receptors stably expressed in membranes of CHO cells (K(d)=0.13+/-0.05 nM; B(max)=2.99+/-0.60 pmol/mg protein): the B(max) was double that of [(3)H]-8-OH-DPAT. GppNHp strongly decreased [(3)H]-8-OH-DPAT binding but scarcely changed [(3)H]-S 14506 binding; calcium, magnesium and manganese had little effect on [(3)H]-S 14506 binding in CHO cells. Antagonists (WAY 100635, WAY 100135) and inverse agonists (spiperone and metitepine) displaced [(3)H]-S 14506 binding with high affinity and Hill slopes close to unity, whereas agonists (5-HT and 5-CT) displayed low affinity with low Hill slopes: partial agonists (buspirone, ipsapirone) showed intermediate properties. In fusion proteins of h 5-HT(1A) receptors with G(ialpha1) the compound potently increased high-affinity GTPase, with a steeper Hill slope than for 5-HT, which may indicate positive cooperativity. The maximum response for S 14506 in these assays was equivalent to 5-HT, indicating it to be a full agonist.In molecular modelling studies, using a three-site model of the 5-HT(1A) receptor, S 14506 spanned between the 5-HT recognition site and the "arginine switch" (DRY microdomain) postulated to activate the interaction of the receptor with the G protein. Thus it is possible to synthesise ligands at G-protein-coupled receptors which are highly potent agonists, but which are structurally related to inverse agonists and show some features of antagonist/inverse agonist binding.
Neuropharmacology 04/2001; 40(3):334-44. · 4.81 Impact Factor
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ABSTRACT: To assess the relative capacity of the human delta opioid receptor to activate closely related G proteins, fusion proteins were constructed in which the alpha-subunits of either G(i1) or G(o1), containing point mutations to render them insensitive to the actions of pertussis toxin, were linked in-frame with the C-terminus of the receptor. Following transient and stable expression in HEK 293 cells, both constructs bound the antagonist [(3)H]naltrindole with high affinity. D-ala(2),D-leu(5) Enkephalin effectively inhibited forskolin-stimulated adenylyl cyclase activity in intact cells in a concentration-dependent, but pertussis toxin-insensitive, manner. The high-affinity GTPase activity of both constructs was also stimulated by D-ala(2),D-leu(5) enkephalin with similar potency. However, enzyme kinetic analysis of agonist stimulation of GTPase activity demonstrated that the GTP turnover number produced in response to D-ala(2),D-leu(5) enkephalin was more than three times greater for G(i1)alpha than for G(o1)alpha. As the effect of agonist in both cases was to increase V:(max) without increasing the observed K:(m) for GTP, this is consistent with receptor promoting greater guanine nucleotide exchange, and thus activation, of G(i1)alpha compared with G(o1)alpha. An equivalent fusion protein between the human mu opioid receptor-1 and G(i1)alpha produced a similar D-ala(2),D-leu(5) enkephalin-induced GTP turnover number as the delta opioid receptor-G(i1)alpha fusion construct, consistent with agonist occupation of these two opioid receptor subtypes being equally efficiently coupled to activation of G(i1)alpha.
Journal of Neurochemistry 04/2001; 76(6):1805-13. · 4.06 Impact Factor
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ABSTRACT: Coexpression of the rat thyrotropin releasing hormone receptor-1 with beta-arrestin 1-green fluorescent protein (GFP) in human embryonic kidney 293 cells results in agonist-dependent translocation of the arrestin to the plasma membrane followed by its cointernalization with the receptor. Truncations of the receptor C-terminal tail from 93 to 50 amino acids did not alter this. Truncations to fewer than 47 amino acids prevented such interactions and inhibited but did not fully eliminate agonist-induced internalization of the receptor. Deletion and site-directed mutants of the C-terminal tail indicated that separate elimination of a potential casein kinase II phosphorylation site or clathrin/clathrin adapter motifs was insufficient to prevent either internalization of the receptor or its cointernalization with beta-arrestin 1-GFP. Alteration of sites of acylation reduced internalization and prevented interactions with beta-arrestin 1-GFP. Combinations of these mutants resulted in lack of interaction with beta-arrestin 1-GFP and a 10-fold reduction in internalization of the receptor. Despite this, the receptor construct that lacked the three protein sequence motifs was fully functional. These studies map sites that contribute the interactions of the thyrotropin releasing hormone receptor-1 C-terminal tail required for effective contacts with beta-arrestin 1-GFP and indicate key roles for these interactions in agonist-induced internalization of the receptor.
Molecular Pharmacology 03/2001; 59(2):375-85. · 4.88 Impact Factor
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Trends in Pharmacological Sciences 11/2000; 21(10):362-3. · 10.93 Impact Factor
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ABSTRACT: During the past 10 years or so, associated with the introduction of molecular biology techniques to G protein-coupled receptor (GPCR) research, outstanding progress has been made in understanding the mechanisms of action of these key proteins and their physiological functions. in-vivo manipulation of levels of GPCRs using transgenic and gene knock-out approaches have been particularly successful in assessing the roles of specific GPCRs in animal physiology. Drug discovery is aiming to produce highly specific compounds based on subtle definition of receptor subtypes which can best be studied using heterologous expression of wild type or mutated forms of cDNA or genes encoding these proteins. Furthermore, new therapeutic opportunities may be provided by investigation of orphan receptors, the natural ligands for which remain unidentified. Some human diseases have been shown to be associated with rare mutations of GPCRs and the possibility that widely distributed polymorphisms in GPCR genes may allow selective therapeutic strategies for population subgroups is driving the development of the science of pharmacogenetics.
Current Medicinal Chemistry 10/2000; 7(9):889-96. · 4.86 Impact Factor
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ABSTRACT: Agonist-stimulated high affinity GTPase activity of fusion proteins between the alpha(2A)-adrenoreceptor and the alpha subunits of forms of the G proteins G(i1), G(i2), G(i3), and G(o1), modified to render them insensitive to the action of pertussis toxin, was measured following transient expression in COS-7 cells. Addition of a recombinant regulator of G protein signaling protein, RGS4, did not significantly affect basal GTPase activity nor agonist stimulation of the fusion proteins containing Galpha(i1) and Galpha(i3) but markedly enhanced agonist-stimulation of the proteins containing Galpha(i2) and Galpha(o1.) The effect of RGS4 on the alpha(2A)-adrenoreceptor-Galpha(o1) fusion protein was concentration-dependent with EC(50) of 30 +/- 3 nm and the potency of the receptor agonist UK14304 was reduced 3-fold by 100 nm RGS4. Equivalent reconstitution with Asn(88)-Ser RGS4 failed to enhance agonist function on the alpha(2A)-adrenoreceptor-Galpha(o1) or alpha(2A)-adrenoreceptor-Galpha(i2) fusion proteins. Enzyme kinetic analysis of the GTPase activity of the alpha(2A)-adrenoreceptor-Galpha(o1) and alpha(2A)-adrenoreceptor-Galpha(i2) fusion proteins demonstrated that RGS4 both substantially increased GTPase V(max) and significantly increased K(m) of the fusion proteins for GTP. The increase in K(m) for GTP was dependent upon RGS4 amount and is consistent with previously proposed mechanisms of RGS function. Agonist-stimulated GTPase turnover number in the presence of 100 nm RGS4 was substantially higher for alpha(2A)-adrenoreceptor-Galpha(o1) than for alpha(2A)-adrenoreceptor-Galpha(i2). These studies demonstrate that although RGS4 has been described as a generic stimulator of the GTPase activity of G(i)-family G proteins, selectivity of this interaction and quantitative variation in its function can be monitored in the presence of receptor activation of the G proteins.
Journal of Biological Chemistry 09/2000; 275(31):23693-9. · 4.77 Impact Factor
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ABSTRACT: Constitutively active forms of the hamster alpha(1b)-adrenoceptor can be produced from the point mutations Asp(142)Ala or Ala(293)Glu or exchange of a small segment of the third intracellular loop with the equivalent region of the beta(2)-adrenoceptor. Green fluorescent protein (GFP)-tagged forms of each of these mutants and of the wild type alpha(1b)-adrenoceptor were expressed stably in HEK293 cells. The wild type alpha(1b)-adrenoceptor-GFP was expressed both at the plasma membrane and with a distinctly perinuclear punctate pattern. Sustained treatment with a range of antagonist/inverse agonist ligands failed to modulate the cellular distribution or levels of expression of this construct. The form of the alpha(1b)-adrenoceptor containing the beta(2)-adrenoceptor sequence substitution was predominantly located in punctate intracellular vesicles and sustained challenge with the same series of antagonists/inverse agonists produced a 5-fold up-regulation of protein levels with elevation of both plasma membrane and intracellular receptor. Quantification of these effects could be produced by spectrofluorometric analysis of cells grown in a 96-well microtiter plate. In contrast, both the Asp(142)Ala and Ala(293)Glu forms of the alpha(1b)-adrenoceptor-GFP were located predominantly at the plasma membrane. Levels of these two point mutants were not increased by any of the antagonist/inverse agonist ligands tested, although the sequence substitution mutation encompasses codon 293. Resolution of constitutive activity and ligand-induced up-regulation was further exemplified by a mutant lacking eight serine residues in the C-terminal tail that displayed little constitutive activity but was up-regulated by sustained ligand challenge. These results demonstrate the nonequivalence of mutations in their regulation by antagonist/inverse agonist ligands.
Molecular Pharmacology 09/2000; 58(2):438-48. · 4.88 Impact Factor
