The G protein-coupled receptor rhodopsin in the native membrane

The higher-order structure of G protein-coupled receptors (GPCRs) in membranes may involve dimerization and formation of even larger oligomeric complexes. Here, we have investigated the organization of the prototypical GPCR rhodopsin in its native membrane by electron and atomic force microscopy (AFM). Disc membranes from mice were isolated and observed by AFM at room temperature. In all experimental conditions, rhodopsin forms structural dimers organized in paracrystalline arrays. A semi-empirical molecular model for the rhodopsin paracrystal is presented validating our previously reported results. Finally, we compare our model with other currently available models describing the supramolecular structure of GPCRs in the membrane.     

Evidence for a G protein-coupled gamma-hydroxybutyric acid receptor

gamma-Hydroxybutyric acid (GHB) is a naturally occurring metabolite of GABA that has been postulated to exert ubiquitous neuropharmacological effects through GABA(B) receptor (GABA(B)R)-mediated mechanisms. The alternative hypothesis that GHB acts via a GHB-specific, G protein-coupled presynaptic receptor that is different from the GABA(B)R was tested. The effect of GHB on regional and subcellular brain adenylyl cyclase in adult and developing rats was determined and compared with that of the GABA(B)R agonist (-)-baclofen. Also, using guanosine 5'-O:-(3-[(35)S]thiotriphosphate) ([(35)S]GTPgammaS) binding and low-K:(m) GTPase activity as markers the effects of GHB and (-)-baclofen on G protein activity in the brain were determined. Neither GHB nor baclofen had an effect on basal cyclic AMP (cAMP) levels. GHB significantly decreased forskolin-stimulated cAMP levels by 40-50% in cortex and hippocampus but not thalamus or cerebellum, whereas (-)-baclofen had an effect throughout the brain. The effect of GHB on adenylyl cyclase was observed in presynaptic and not postsynaptic subcellular tissue preparations, but the effect of baclofen was observed in both subcellular preparations. The GHB-induced alteration in forskolin-induced cAMP formation was blocked by a specific GHB antagonist but not a specific GABA(B)R antagonist. The (-)-baclofen-induced alteration in forskolin-induced cAMP formation was blocked by a specific GABA(B)R antagonist but not a specific GHB antagonist. The negative coupling of GHB to adenylyl cyclase appeared at postnatal day 21, a developmental time point that is concordant with the developmental appearance of [(3)H]GHB binding in cerebral cortex, but the effects of (-)-baclofen were present by postnatal day 14. GHB and baclofen both stimulated [(35)S]GTPgammaS binding and low-K:(m) GTPase activity by 40-50%. The GHB-induced effect was blocked by GHB antagonists but not by GABA(B)R antagonists and was seen only in cortex and hippocampus. The (-)-baclofen-induced effect was blocked by GABA(B)R antagonists but not by GHB antagonists and was observed throughout the brain. These data support the hypothesis that GHB induces a G protein-mediated decrease in adenylyl cyclase via a GHB-specific G protein-coupled presynaptic receptor that is different from the GABA(B)R.     

Biosynthesis and biophysical analysis of domains of a yeast G protein-coupled receptor

The alpha-factor receptor(Ste2p) is required for the sexual conjugation of the yeast Saccharomyces cerevisiae. Ste2p belongs to the G protein-coupled receptor (GPCR) family sharing a common heptahelical transmembrane structure. Biological synthesis of regions of Ste2p fused to a leader protein in Escherichia coli yielded milligram quantities of polypeptides that corresponded to one or two transmembrane domains. Fusion proteins were characterized by polyacrylamide gel electrophoresis, high performance liquid chromatography, and mass spectrometry. CD studies on the fusion proteins in trifluoroethanol:water mixtures indicated the existence of alpha-helical structures in the single- and the double-transmembrane domains. NMR experiments were performed in CDCl(3):CD(3)OH:H(2)O (4:4:1) on the (15)N-labeled single-transmembrane peptide showing a clear dispersion of the nitrogen-amide proton correlation cross peaks indicative of a high-purity, uniformly labeled molecule. The results indicate that single- and double-transmembrane domains of a GPCR can be produced by biosynthetic methods in quantities and purity sufficient for biophysical studies.     

Study of the binding environment of alpha-factor in its G protein-coupled receptor using fluorescence spectroscopy.

Mating in Saccharomyces cerevisiae is induced by the interaction of alpha-factor (W1H2W3L4Q5L6K7P8G9Q10P11M12Y13) with its cognate G protein-coupled receptor (Ste2p). Fifteen fluorescently labeled analogs of alpha-factor in which the 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) group was placed at the alphaN-terminus and in side-chains at positions 1, 3, 4, 6, 7, 12 and 13 were synthesized and assayed for biological activity and receptor affinity. Eleven of the analogs retained 6-60% of the biological activity of the alpha-factor, as judged using a growth arrest assay. The binding affinities depended on the position of NBD attachment in the peptide and the distance of the tag from the backbone. Derivatization of the positions 3 and 7 side-chains with the NBD group resulted in analogs with affinities of 17-35% compared with that of alpha-factor. None of the other NBD-containing agonists had sufficient receptor affinity or strong enough emission for fluorescence analysis. The position 3 and 7 analogs were investigated using fluorescence spectroscopy and collisional quenching by KI in the presence of Ste2p in yeast membranes. The results showed that the lambda max of NBD in the position 7 side-chain shifted markedly to the blue (510 nm) when separated by 4 or 6 bonds from the peptide backbone and that this probe was shielded from quenching by KI. In contrast, separation by 3, 5, 10 or more bonds resulted in lambda max ( approximately 540 nm) and collisional quenching constants consistent with increasing degrees of exposure. The NBD group in the position 3 side-chain was also found to be blue shifted (lambda max=520 nm) and shielded from solvent. These results indicate that the position 7 side-chain is likely interacting with a pocket formed by extracellular domains of Ste2p, whereas the side-chain of Trp3 is in a hydrophobic pocket possibly within the transmembrane region of the receptor.     

Expression and biophysical analysis of a triple-transmembrane domain-containing fragment from a yeast G protein-coupled receptor

Structural characterization of G protein-coupled receptors (GPCRs) is hindered by the inherent hydrophobicity, flexibility, and large size of these signaling proteins. Insights into conformational preferences and the three-dimensional (3D) structure of domains of these receptors can be obtained using polypeptide fragments of these proteins. Herein, we report the expression, purification, and biophysical characterization of a three-transmembrane domain-containing 131-residue fragment of the yeast α-factor receptor, Ste2p. Ste2p TM1–TM3 (G31–R161) was expressed as a TrpΔLE fusion protein in Escherichia coli. The expressed protein was subject to CNBr cleavage to remove the fusion tag and TM1–TM3 was purified by reverse-phased HPLC. The cleavage product was isolated in yields of up to 20 mg per liter of culture in both unlabeled and uniformly [15N]-labeled and [15N, 13C, 2H]-labeled forms. The secondary structure of TM1–TM3 was determined to be helical in a number of membrane mimetic environments, including 2,2,2-trifluoroethanol (TFE):water and lysomyristoylphosphatidylglycerol (LMPG) detergent micelles by circular dichroism. Preliminary HSQC analysis in 50% TFE:water and LMPG micelles prepared in sodium phosphate and 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES) buffers revealed that this fragment is suitable for structural analysis by nuclear magnetic resonance (NMR). Complete backbone assignments and a detailed localization of the secondary structural elements of TM1–TM3 in 50% TFE:water have been achieved.     

Kynurenic acid as a ligand for orphan G protein-coupled receptor GPR35

Local catabolism of the essential amino acid tryptophan is considered an important mechanism in regulating immunological and neurological responses. The kynurenine pathway is the main route for the non-protein metabolism of tryptophan. The intermediates of the kynurenine pathway are present at micromolar concentrations in blood and are regulated by inflammatory stimuli. Here we show that GPR35, a previously orphan G protein-coupled receptor, functions as a receptor for the kynurenine pathway intermediate kynurenic acid. Kynurenic acid elicits calcium mobilization and inositol phosphate production in a GPR35-dependent manner in the presence of G(qi/o) chimeric G proteins. Kynurenic acid stimulates [35S]guanosine 5'-O-(3-thiotriphosphate) binding in GPR35-expressing cells, an effect abolished by pertussis toxin treatment. Kynurenic acid also induces the internalization of GPR35. Expression analysis indicates that GPR35 is predominantly detected in immune cells and the gastrointestinal tract. Furthermore, we show that kynurenic acid inhibits lipopolysaccharide-induced tumor necrosis factor-alpha secretion in peripheral blood mononuclear cells. Our results suggest unexpected signaling functions for kynurenic acid through GPR35 activation.     

Unnatural amino acid incorporation into virus-like particles

Virus-like particles composed of hepatitis B virus (HBV) or bacteriophage Qbeta capsid proteins have been labeled with azide- or alkyne-containing unnatural amino acids by expression in a methionine auxotrophic strain of E. coli. The substitution does not affect the ability of the particles to self-assemble into icosahedral structures indistinguishable from native forms. The azide and alkyne groups were addressed by Cu(I)-catalyzed [3 + 2] cycloaddition: HBV particles were decomposed by the formation of more than 120 triazole linkages per capsid in a location-dependent manner, whereas Qbeta suffered no such instability. The marriage of these well-known techniques of sense-codon reassignment and bioorthogonal chemical coupling provides the capability to construct polyvalent particles displaying a wide variety of functional groups with near-perfect control of spacing.     

G protein-coupled receptor systems and their lipid environment in health disorders during aging

Cells, tissues and organs undergo phenotypic changes and deteriorate as they age. Cell growth arrest and hyporesponsiveness to extrinsic stimuli are all hallmarks of senescent cells. Most such external stimuli received by a cell are processed by two different cell membrane systems: receptor tyrosine kinases (RTKs) and G protein-coupled receptors (GPCRs). GPCRs form the largest gene family in the human genome and they are involved in most relevant physiological functions. Given the changes observed in the expression and activity of GPCRs during aging, it is possible that these receptors are directly involved in aging and certain age-related pathologies. On the other hand, both GPCRs and G proteins are associated with the plasma membrane and since lipid-protein interactions regulate their activity, they can both be considered to be sensitive to the lipid environment. Changes in membrane lipid composition and structure have been described in aged cells and furthermore, these membrane changes have been associated with alterations in GPCR mediated signaling in some of the main health disorders in elderly subjects. Although senescence could be considered a physiologic process, not all aging humans develop the same health disorders. Here, we review the involvement of GPCRs and their lipid environment in the development of the major human pathologies associated with aging such as cancer, neurodegenerative disorders and cardiovascular pathologies.     

Expression and biophysical analysis of two double-transmembrane domain-containing fragments from a yeast G protein-coupled receptor

Fragments of G protein-coupled receptors (GPCRs) are widely used as models to investigate these polytopic integral-membrane, signal-transducing molecules, but have proven difficult to prepare in quantities necessary for NMR analyses. We report on the biosynthesis of two double transmembrane (TM) containing fragments of Ste2p, the alpha-factor GPCR from the yeast Saccharomyces cerevisiae. Ste2p(G31-T110) [TM1-TM2] and Ste2p(R231-S339) [TM6-TM7-CT40] were expressed as TrpDeltaLE fusion proteins in Escherichia coli and released by CNBr cleavage. Expression yields were optimized using different strains and induction parameters, and by performing CNBr cleavage directly on inclusion bodies. Nonlabeled and uniformly labeled [15N]-TM1-TM2 and TM6-TM7-CT40, as well as uniformly labeled [15N,13C]-TM1-TM2 and TM1-TM2 selectively labeled with [15N-Ala], [15N-Phe], [15N-Leu], [15N-Ile], and [15N-Val] were prepared. Yields of target peptides with >95% homogeneity varied from 3 mg/L of fermentation ([15N]-TM6-TM7-CT40) to 20 mg/L (selectively labeled TM1-TM2). The high level biosynthesis and the efficient CNBr processing and purification yields allowed the initiation of a comprehensive biophysical analysis of TM1-TM2 and TM6-TM7-CT40. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis showed that TM1-TM2 was monomeric in this micellar environment, whereas TM6-TM7-CT40 migrated as a dimer. CD analysis indicated that TM1-TM2 was highly helical in SDS and 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-RAC-(1-glycerol)], but had a tendency to aggregate in dodecylphosphocholine micelles. Similar results were found with TM6-TM7-CT40. Conditions for NMR measurements were optimized, and both TM1-TM2 and TM6-TM7-CT40 exhibited more than 90% of the expected crosspeaks in the [15N,1H]-HSQC spectrum. These findings set the stage for the determination of the 3D structure of these large domains of a GPCR in micelles using high-resolution NMR.     

植物G蛋白偶联受体及其在信号转导中的作用

G蛋白偶联受体(G protein-coupled receptors,GPCRs)是具有7个跨膜螺旋的蛋白质受体,是人体内最大的蛋白质超家族.GPCRs能调控细胞周期,参与多种植物信号通路以及影响一系列的代谢和分化活动.简要介绍了GPCR和G蛋白介导的信号转导机制,GPCRs的结构和植物GPCR及其在植物跨膜信号转导中的作用,并对GPCR的信号转导机制及植物抗病反应分子机制的研究提出展望.     

Identification of a novel sequence mediating regulated endocytosis of the G protein-coupled alpha-pheromone receptor in yeast.

The Saccharomyces cerevisiae alpha-pheromone receptor, a polytopic, G protein-coupled, membrane protein, is internalized after binding of alpha-factor. Mutational analysis suggested that the first 39 residues of the receptor's cytoplasmic tail carries sufficient information for internalization. A point mutation in one of these 39 residues, K337 to R337, renders the receptor nonfunctional for endocytosis. Other residues, D335 and S338, contribute to the efficiency of internalization. When the sequence DAKSS is added onto a severely truncated receptor, endocytosis of the receptor is restored, showing that this sequence functions to mediate or to signal interaction with the endocytic machinery. Analysis of pheromone response and recovery in strains expressing mutant receptors suggests that receptor internalization is not important for response but contributes to recovery from pheromone.     

When a G protein-coupled receptor does not couple to a G protein

Classically, G protein-coupled receptors (GPCRs) relay signals by directly activating heterotrimeric guanine nucleotide-binding proteins (G proteins). Increasing evidence indicates that GPCRs may also signal through G protein-independent pathways. JAK/STATs, Src-family tyrosine kinases, GRKs/beta-arrestins, and PDZ domain-containing proteins have been suggested to directly relay signals from GPCRs independent of G proteins. In addition, our laboratory recently reported that the beta(2) adrenergic receptor (beta(2)AR) could switch from G protein-coupled to G protein-independent ERK (extracellular signal-regulated kinase) activation in an agonist dosage-dependent manner. This finding provides a novel mechanism for G protein-independent GPCR signaling. This review focuses on recent progress in understanding the mechanisms by which G protein-independent GPCR signaling occurs.     

Conformation of a double-membrane-spanning fragment of a G protein-coupled receptor: Effects of hydrophobic environment and pH

Overcoming the problems associated with the expression, purification and in vitro handling of membrane proteins requires an understanding of the factors governing the folding and stability of such proteins in detergent solutions. As a sequel to our earlier report (Biochim. Biophys. Acta 1747(2005), 133-140), we describe an improved purification procedure and a detailed structural analysis of a fragment of the μ-opioid receptor (‘TM2-3’) that comprises the second and third transmembrane segments and the extracellular loop that connects them. Circular dichroism (CD) spectroscopy of TM2-3 in 2,2,2-trifluoroethanol gave a helical content similar to that predicted by published homology models, while spectra acquired in several detergents showed significantly lower helical contents. This indicates that this part of the μ-opioid receptor has an intrinsic propensity to be highly helical in membrane-like environments, but that in detergent solutions, this helical structure is not fully formed. Proteolysis of TM2-3 with trypsin showed that the helical portions of TM2 and TM3 are both shorter than their predicted lengths, indicating that helix-helix interactions in the full-length receptor are apparently important for stabilizing their conformation. Lengthening the alkyl chain of the detergent led to a small but significant increase in the helicity of TM2-3, suggesting that hydrophobic mismatch could play an important role in the stabilization of transmembrane helices by detergents. Protonation of aspartic acid residues in detergent-solubilized TM2-3 also caused a significant increase in helicity. Our results thus suggest that detergent alkyl chain-length and pH may influence membrane protein stability by modulating the stability of individual transmembrane segments.     

Site-specific incorporation of keto amino acids into functional G protein-coupled receptors using unnatural amino acid mutagenesis

G protein-coupled receptors (GPCRs) are ubiquitous heptahelical transmembrane proteins involved in a wide variety of signaling pathways. The work described here on application of unnatural amino acid mutagenesis to two GPCRs, the chemokine receptor CCR5 (a major co-receptor for the human immunodeficiency virus) and rhodopsin (the visual photoreceptor), adds a new dimension to studies of GPCRs. We incorporated the unnatural amino acids p-acetyl-L-phenylalanine (Acp) and p-benzoyl-L-phenylalanine (Bzp) into CCR5 at high efficiency in mammalian cells to produce functional receptors harboring reactive keto groups at three specific positions. We obtained functional mutant CCR5, at levels up to approximately 50% of wild type as judged by immunoblotting, cell surface expression, and ligand-dependent calcium flux. Rhodopsin containing Acp at three different sites was also purified in high yield (0.5-2 microg/10(7) cells) and reacted with fluorescein hydrazide in vitro to produce fluorescently labeled rhodopsin. The incorporation of reactive keto groups such as Acp or Bzp into GPCRs allows their reaction with different reagents to introduce a variety of spectroscopic and other probes. Bzp also provides the possibility of photo-cross-linking to identify precise sites of protein-protein interactions, including GPCR binding to G proteins and arrestins, and for understanding the molecular basis of ligand recognition by chemokine receptors.     

A G protein-coupled receptor for UDP-glucose

Uridine 5'-diphosphoglucose (UDP-glucose) has a well established biochemical role as a glycosyl donor in the enzymatic biosynthesis of carbohydrates. It is less well known that UDP-glucose may possess pharmacological activity, suggesting that a receptor for this molecule may exist. Here, we show that UDP-glucose, and some closely related molecules, potently activate the orphan G protein-coupled receptor KIAA0001 heterologously expressed in yeast or mammalian cells. Nucleotides known to activate P2Y receptors were inactive, indicating the distinctly novel pharmacology of this receptor. The receptor is expressed in a wide variety of human tissues, including many regions of the brain. These data suggest that some sugar-nucleotides may serve important physiological roles as extracellular signaling molecules in addition to their familiar role in intermediary metabolism.     

Conformation of a double-membrane-spanning fragment of a G protein-coupled receptor: effects of hydrophobic environment and pH

Overcoming the problems associated with the expression, purification and in vitro handling of membrane proteins requires an understanding of the factors governing the folding and stability of such proteins in detergent solutions. As a sequel to our earlier report (Biochim. Biophys. Acta 1747(2005), 133-140), we describe an improved purification procedure and a detailed structural analysis of a fragment of the mu-opioid receptor ('TM2-3') that comprises the second and third transmembrane segments and the extracellular loop that connects them. Circular dichroism (CD) spectroscopy of TM2-3 in 2,2,2-trifluoroethanol gave a helical content similar to that predicted by published homology models, while spectra acquired in several detergents showed significantly lower helical contents. This indicates that this part of the mu-opioid receptor has an intrinsic propensity to be highly helical in membrane-like environments, but that in detergent solutions, this helical structure is not fully formed. Proteolysis of TM2-3 with trypsin showed that the helical portions of TM2 and TM3 are both shorter than their predicted lengths, indicating that helix-helix interactions in the full-length receptor are apparently important for stabilizing their conformation. Lengthening the alkyl chain of the detergent led to a small but significant increase in the helicity of TM2-3, suggesting that hydrophobic mismatch could play an important role in the stabilization of transmembrane helices by detergents. Protonation of aspartic acid residues in detergent-solubilized TM2-3 also caused a significant increase in helicity. Our results thus suggest that detergent alkyl chain-length and pH may influence membrane protein stability by modulating the stability of individual transmembrane segments.     

Molecular basis of inverse agonism in a G protein-coupled receptor

G protein-coupled receptors (GPCRs) recognize a wide variety of extracellular ligands to control diverse physiological processes. Compounds that bind to such receptors can either stimulate, fully or partially (full or partial agonists), or reduce (inverse agonists) the receptors' basal activity and receptor-mediated signaling. Various studies have shown that the activation of receptors through binding of agonists proceeds by conformational changes as the receptor switches from a resting to an active state leading to G protein signaling. Yet the molecular basis for differences between agonists and inverse agonists is unclear. These different classes of compounds are assumed to switch the receptors' conformation in distinct ways. It is not known, however, whether such switching occurs along a linear 'on-off' scale or whether agonists and inverse agonists induce different switch mechanisms. Using a fluorescence-based approach to study the alpha2A-adrenergic receptor (alpha(2A)AR), we show that inverse agonists are differentiated from agonists in that they trigger a very distinct mode of a receptor's switch. This switch couples inverse agonist binding to the suppression of activity in the receptor.     

C5a receptor oligomerization. II. Fluorescence resonance energy transfer studies of a human G protein-coupled receptor expressed in yeast

Recent studies demonstrate that members of the superfamily of G protein-coupled receptors (GPCRs) form oligomers both in vitro and in vivo. The mechanisms by which GPCRs oligomerize and the roles of accessory proteins in this process are not well understood. We used disulfide-trapping experiments to show that C5a receptors, expressed in mammalian cells, reside in membranes as oligomers (Klco, J. M., Lassere, T. B., and Baranski, T. J. (2003) J. Biol. Chem. 278, 35345-35353). To begin to address how C5a receptors form oligomers, we now use fluorescence resonance energy transfer experiments on human C5a receptors expressed in the lower eukaryote Saccharomyces cerevisiae. C5a receptors tagged with variants of the green fluorescent protein display energy transfer in intact yeast, demonstrating that mammalian accessory proteins are not required for C5a receptor oligomerization. In both intact yeast cells and membrane preparations, agonist does not affect FRET efficiency, and little energy transfer is observed between the C5a receptor and a co-expressed yeast pheromone receptor (encoded by STE2), indicating that C5a receptor oligomerization is both receptor-specific and constitutive. FRET studies performed on fractionated membranes demonstrate similar levels of energy transfer between tagged C5a receptors in endoplasmic reticulum compared with plasma membrane, and urea washing of membranes has little effect on the extent of energy transfer. The oligomerization of C5a receptors expressed in yeast displays characteristics similar to those observed for other GPCRs studied in mammalian cells. This model system should prove useful for further studies to define mechanisms of oligomerization of mammalian GPCRs.     

Changes in G protein-coupled receptor sorting protein affinity regulate postendocytic targeting of G protein-coupled receptors

After activation, most G protein-coupled receptors (GPCRs) are regulated by a cascade of events involving desensitization and endocytosis. Internalized receptors can then be recycled to the plasma membrane, retained in an endosomal compartment, or targeted for degradation. The GPCR-associated sorting protein, GASP, has been shown to preferentially sort a number of native GPCRs to the lysosome for degradation after endocytosis. Here we show that a mutant beta(2) adrenergic receptor and a mutant mu opioid receptor that have previously been described as lacking "recycling signals" due to mutations in their C termini in fact bind to GASP and are targeted for degradation. We also show that a mutant dopamine D1 receptor, which has likewise been described as lacking a recycling signal, does not bind to GASP and is therefore not targeted for degradation. Together, these results indicate that alteration of receptors in their C termini can expose determinants with affinity for GASP binding and consequently target receptors for degradation.