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PubMed2003

Constitutive desensitization: a new paradigm for g protein-coupled receptor regulation.

GPCRs are a large family of cell-surface proteins that regulate many important biochemical pathways and physiological responses. The isolation and characterization of GPCRs represent one of the more remarkable success stories that occurred during the revolution in biology of the last quarter century. Of the many discoveries that originated in the laboratory of Robert Lefkowitz at Duke University concerning GPCR regulation, none is more fundamental than the elucidation of the families of GRKs and arrestin proteins that terminate GPCR signaling. In this essay, we will discuss how advances in microscopy and biology have made the visualization of GPCR, GRK, and arrestin activity possible in single cells. Additionally, we will discuss how imaging studies using arrestins and a naturally occurring mutant of the vasopressin receptor led to the recognition of a novel phenotypic receptor behavior, in which the receptor desensitizes in the absence of agonist. We have termed this process constitutive desensitization, and this unexpected receptor property suggests that it may be possible to develop novel classes of signal-inhibiting drugs distinct from conventional antagonists.

Down-Regulation↗
PubMed2004

Characterization of CHO cells stably expressing a G alpha 16/z chimera for high throughput screening of GPCRs.

G protein-coupled receptors (GPCRs) are important therapeutic targets for drug discovery. The identification and characterization of new ligands ideally requires the use of high throughput assays that are applicable to all GPCR subtypes. To circumvent the problem of different GPCRs coupling to distinct intracellular second messenger pathways, we describe a new method that uses the chimeric Galpha protein 16z25 to facilitate this process. Stably expressed in Chinese hamster ovary cells, 16z25 allows G(i/o)- and G(s)-coupled receptors to mobilize intracellular Ca(2+) upon agonist stimulation. We have generated nine cell lines each stably expressing 16z25 and a GPCR. All cell lines respond to appropriate agonist stimulation in fluorometric imaging plate reader (FLIPR) assays with robust and potent Ca(2+) mobilization. Several of these lines have been pharmacologically characterized using agonists and antagonists. We also demonstrate that the coexpression of GPCR and 16z25 does not interfere with the receptors' ability to activate endogenous signaling pathways. The ability of 16z25 to functionally mediate the agonist stimulation of a broad spectrum of GPCRs indicates that the use of cell lines stably coexpressing this chimera and GPCRs will simplify the drug screening process and aid in the deorphanization of new receptors.

Amino Acid Sequence↗
PubMed2005

Galpha subunit Gpa2 recruits kelch repeat subunits that inhibit receptor-G protein coupling during cAMP-induced dimorphic transitions in Saccharomyces cerevisiae.

All eukaryotic cells sense extracellular stimuli and activate intracellular signaling cascades via G protein-coupled receptors (GPCR) and associated heterotrimeric G proteins. The Saccharomyces cerevisiae GPCR Gpr1 and associated Galpha subunit Gpa2 sense extracellular carbon sources (including glucose) to govern filamentous growth. In contrast to conventional Galpha subunits, Gpa2 forms an atypical G protein complex with the kelch repeat Gbeta mimic proteins Gpb1 and Gpb2. Gpb1/2 negatively regulate cAMP signaling by inhibiting Gpa2 and an as yet unidentified target. Here we show that Gpa2 requires lipid modifications of its N-terminus for membrane localization but association with the Gpr1 receptor or Gpb1/2 subunits is dispensable for membrane targeting. Instead, Gpa2 promotes membrane localization of its associated Gbeta mimic subunit Gpb2. We also show that the Gpa2 N-terminus binds both to Gpb2 and to the C-terminal tail of the Gpr1 receptor and that Gpb1/2 binding interferes with Gpr1 receptor coupling to Gpa2. Our studies invoke novel mechanisms involving GPCR-G protein modules that may be conserved in multicellular eukaryotes.

Amino Acid Sequence↗
PubMed2006

Cooperative mitogenic signaling by G protein-coupled receptors and growth factors is dependent on G(q/11).

Previously we reported that the G protein-coupled receptor (GPCR) agonist thrombin potentiated the mitogenic effect of epidermal growth factor (EGF) on human airway smooth muscle (ASM) by promoting sustained late-phase activation of PI3K and p70S6K via a pathway dependent on Gbetagamma subunits of heterotrimeric G proteins. Here, we provide additional mechanistic insight and reveal the robustness of this phenomenon by demonstrating that H1 histamine and thromboxane receptors utilize the same mechanism to augment ASM growth via specific activation of the heterotrimeric G protein G(q/11). Thrombin, histamine, and U46619 all enhanced EGF-stimulated [3H]-thymidine incorporation as well as late-phase Akt and p70S6K phosphorylation in ASM cultures. Heterologous expression of Gbetagamma sequestrants (GRK2CT-GFP or Galpha(i)G203A), as well as GRK2NT-GFP (an RGS protein for G(q/11)) but neither p115RhoGEFRGS-GFP (an RGS for G(12/13)) nor pertussis toxin pretreatment (inactivating G(i/o)), attenuated the effects on both signaling and growth. Inhibition of Rho, Rho kinase, or Src, or modulation of arrestin expression did not significantly affect the cooperative signaling by EGF and any of the GPCR agonists. Thus, G(q/11)-coupled receptors are the principal GPCR subfamily mediating cooperative mitogenic signaling in ASM, acting through Gbetagamma-dependent, and Src/arrestin-independent activation of PI3K and p70S6K.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed2006

Na,K-ATPase alpha1-subunit dephosphorylation by protein phosphatase 2A is necessary for its recruitment to the plasma membrane.

In alveolar epithelial cells, G-protein coupled-receptors agonists (GPCR) induce the recruitment of the Na,K-ATPase to the plasma membrane. Here we report that for the recruitment of the Na,K-ATPase to occur, dephosphorylation of its alpha1-subunit at serine 18 is necessary, as demonstrated by in vitro phosphorylation, mutation of the serine 18 to alanine, and use of a specific phospho-antibody. Several approaches strongly suggest dephosphorylation to be mediated by protein phosphatase 2A (PP2A): 1) Na,K-ATPase dephosphorylation and recruitment were prevented by okadaic acid (OA); 2) the Na,K-ATPase alpha1-subunit is an in vitro substrate for PP2A; and 3) glutathione S-transferase (GST)-fusion proteins binding assays demonstrate a direct interaction between the catalytic subunit of PP2A and the first 90 amino acids of the Na,K-ATPase alpha1-subunit. Finally, GPCR agonists induced a rapid translocation of PP2A from the cytosol to the membrane fraction, which corresponded with increased coimmunoprecipitation and colocalization of PP2A and the Na,K-ATPase. Accordingly, we provide evidence that GPCR agonists promote PP2A translocation to the membrane fraction, leading to the dephosphorylation of the Na,K-ATPase alpha1-subunit at the serine 18 residue and its recruitment to the cell plasma membrane, which is of biological and physiological importance.

Amino Acid Sequence↗
PubMed2006

Enhancing functional production of G protein-coupled receptors in Pichia pastoris to levels required for structural studies via a single expression screen.

We have optimized the expression level of 20 mammalian G protein-coupled receptors (GPCRs) in the methylotrophic yeast Pichia pastoris. We found that altering expression parameters, including growth temperature, and supplementation of the culture medium with specific GPCR ligands, histidine, and DMSO increased the amount of functional receptor, as assessed by ligand binding, by more than eightfold over standard expression conditions. Unexpectedly, we found that the overall amount of GPCR proteins expressed, in most cases, varied only marginally between standard and optimized expression conditions. Accordingly, the optimized expression conditions resulted in a marked fractional increase in the ratio of ligand binding-competent receptor to total expressed receptor. The results of this study suggest a general approach for increasing yields of functional mammalian GPCRs severalfold over standard expression conditions by using a set of optimized expression condition parameters that we have characterized for the Pichia expression system. Overall, we have more than doubled the number of GPCR targets that can be produced in our laboratories in sufficient amounts for structural studies.

Amino Acid Sequence↗
PubMed2002

Classification of G-protein coupled receptors by alignment-independent extraction of principal chemical properties of primary amino acid sequences.

We have developed an alignment-independent method for classification of G-protein coupled receptors (GPCRs) according to the principal chemical properties of their amino acid sequences. The method relies on a multivariate approach where the primary amino acid sequences are translated into vectors based on the principal physicochemical properties of the amino acids and transformation of the data into a uniform matrix by applying a modified autocross-covariance transform. The application of principal component analysis to a data set of 929 class A GPCRs showed a clear separation of the major classes of GPCRs. The application of partial least squares projection to latent structures created a highly valid model (cross-validated correlation coefficient, Q(2) = 0.895) that gave unambiguous classification of the GPCRs in the training set according to their ligand binding class. The model was further validated by external prediction of 535 novel GPCRs not included in the training set. Of the latter, only 14 sequences, confined in rapidly expanding GPCR classes, were mispredicted. Moreover, 90 orphan GPCRs out of 165 were tentatively identified to GPCR ligand binding class. The alignment-independent method could be used to assess the importance of the principal chemical properties of every single amino acid in the protein sequences for their contributions in explaining GPCR family membership. It was then revealed that all amino acids in the unaligned sequences contributed to the classifications, albeit to varying extent; the most important amino acids being those that could also be determined to be conserved by using traditional alignment-based methods.

Amino Acids↗
PubMed2004

Cloning of a novel neuronally expressed orphan G-protein-coupled receptor which is up-regulated by erythropoietin, interacts with microtubule-associated protein 1b and colocalizes with the 5-hydroxytryptamine 2a receptor.

G-protein-coupled receptors (GPCRs) are the largest group of cell surface molecules involved in signal transduction and are receptors for a wide variety of stimuli ranging from light, calcium and odourants to biogenic amines and peptides. It is assumed that systematic genomic data-mining has identified the overwhelming majority of all remaining GPCRs in the genome. Here we report the cloning of a novel orphan GPCR which was identified in a search for erythropoietin-induced genes in the brain as a strongly up-regulated gene. This unknown gene coded for a protein which had a seven-transmembrane topology and key features typical of GPCRs of the A family but a low overall identity to all known GPCRs. The protein, coded ee3, has an unusually high evolutionary conservation and is expressed in neurons in diverse areas of the CNS with relation to integrative functions or motor tasks. A yeast two-hybrid screen for interacting proteins revealed binding to the microtubule-associated protein (MAP) 1b. Coupling to MAP1a has been described for another cognate GPCR, the 5-hydroxytryptamine (5HT) 2a receptor. Surprisingly, we found complete colocalization of ee3 and the 5HT2a receptor. The interaction with MAP1b proved to be critical for the stability or folding of ee3 as in mice lacking MAP1b the ee3 protein was undetectable by immunohistochemistry, although messenger RNA levels remained unchanged. We propose that ee3 is a highly interesting new orphan GPCR with potential connections to erythropoietin and 5HT2a receptor signalling.

Amino Acid Sequence↗
PubMed2005

Melanocortin-1 receptor structure and functional regulation.

The melanogenic actions of the melanocortins are mediated by the melanocortin-1 receptor (MC1R). MC1R is a member of the G-protein-coupled receptors (GPCR) superfamily expressed in cutaneous and hair follicle melanocytes. Activation of MC1R by adrenocorticotrophin or alpha-melanocyte stimulating hormone is positively coupled to the cAMP signaling pathway and leads to a stimulation of melanogenesis and a switch from the synthesis of pheomelanins to the production of eumelanic pigments. The functional behavior of the MC1R agrees with emerging concepts in GPCR signaling including dimerization, coupling to more than one signaling pathway and a high agonist-independent constitutive activity accounting for inverse agonism phenomena. In addition, MC1R displays unique properties such as an unusually high number of natural variants often associated with clearly visible phenotypes and the occurrence of endogenous peptide antagonists. Therefore MC1R is an ideal model to study GPCR function. Here we review our current knowledge of MC1R structure and function, with emphasis on information gathered from the analysis of natural variants. We also discuss recent data on the regulation of MC1R function by paracrine and endocrine factors and by external stimuli such as ultraviolet light.

Amino Acid Sequence↗
PubMed2006

The melanosomal/lysosomal protein OA1 has properties of a G protein-coupled receptor.

The protein product of the ocular albinism type 1 gene, named OA1, is a pigment cell-specific integral membrane glycoprotein, localized to melanosomes and lysosomes and possibly implicated in melanosome biogenesis. Although its function remains unknown, we previously showed that OA1 shares structural similarities with G protein-coupled receptors (GPCRs). To ascertain the molecular function of OA1 and in particular its nature as a GPCR, we adopted a heterologous expression strategy commonly exploited to demonstrate GPCR-mediated signaling in mammalian cells. Here we show that when expressed in COS7 cells OA1 displays a considerable and spontaneous capacity to activate heterotrimeric G proteins and the associated signaling cascade. In contrast, OA1 mutants carrying either a missense mutation or a small deletion in the third cytosolic loop lack this ability. Furthermore, OA1 is phosphorylated and interacts with arrestins, well-established multifunctional adaptors of conformationally active GPCRs. In fact, OA1 colocalizes and coprecipitates with arrestins, which downregulate the signaling of OA1 by specifically reducing its expression levels. These findings indicate that heterologously expressed OA1 exhibits two fundamental properties of GPCRs, being capable to activate heterotrimeric G proteins and to functionally associate with arrestins, and provide proof of principle that OA1 can actually function as a canonical GPCR in mammalian cells.

Amino Acid Sequence↗
PubMed2006

Distinct clathrin-coated pits sort different G protein-coupled receptor cargo.

Upon activation, many G protein-coupled receptors (GPCRs) internalize by clathrin-mediated endocytosis and are subsequently sorted to undergo recycling or lysosomal degradation. Here we observe that sorting can take place much earlier than previously thought, by entry of different GPCRs into distinct populations of clathrin-coated pit (CCP). These distinct populations were revealed by analysis of two purinergic GPCRs, P2Y(1) and P2Y(12), which enter two populations of CCPs in a mutually exclusive manner. The mechanisms underlying early GPCR sorting involve differential kinase-dependent processes because internalization of P2Y(12) is mediated by GPCR kinases (GRKs) and arrestin, whereas P2Y(1) internalization is GRK- and arrestin-independent but requires protein kinase C. Importantly, the beta(2) adrenoceptor which also internalizes in a GRK-dependent manner also traffics exclusively to P2Y(12)-containing CCPs. Our data therefore reveal distinct populations of CCPs that sort GPCR cargo at the plasma membrane using different kinase-dependent mechanisms.

Antibodies, Monoclonal↗
PubMed2005

The study of G-protein coupled receptor oligomerization with computational modeling and bioinformatics.

To achieve a structural context for the analysis of G-protein coupled receptor (GPCR) oligomers, molecular modeling must be used to predict the corresponding interaction interfaces. The task is complicated by the paucity of detailed structural data at atomic resolution, and the large number of possible modes in which the bundles of seven transmembrane (TM) segments of the interacting GPCR monomers can be packed together into dimers and/or higher-order oligomers. Approaches and tools offered by bioinformatics can be used to reduce the complexity of this task and, combined with computational modeling, can serve to yield testable predictions for the structural properties of oligomers. Most of the bioinformatics methods take advantage of the evolutionary relation that exists among GPCRs, as expressed in their sequences and measurable in the common elements of their structural and functional features. These common elements are responsible for the presence of detectable patterns of motifs and correlated mutations evident from the alignment of the sequences of these complex biological systems. The decoding of these patterns in terms of structural and functional determinants can provide indications about the most likely interfaces of dimerization/oligomerization of GPCRs. We review here the main approaches from bioinformatics, enhanced by computational molecular modeling, that have been used to predict likely interfaces of dimerization/oligomerization of GPCRs, and compare results from their application to rhodopsin-like GPCRs. A compilation of the most frequently predicted GPCR oligomerization interfaces points to specific regions of TMs 4-6.

Computational Biology↗
PubMed2006

Fluorophore-assisted light inactivation produces both targeted and collateral effects on N-type calcium channel modulation in rat sympathetic neurons.

Fluorophore-assisted light inactivation (FALI) is a method to inactivate specific proteins on a time scale of seconds to minutes using either diffuse or coherent light. Here we examine a novel FALI modality that utilizes a fluorescein-conjugated polypeptide, alpha-bungarotoxin (BTX) and a 13 amino acid BTX-binding site engineered into the N-terminus of metabotropic glutamate receptor 8a (mGluR8a), a class C G-protein-coupled receptor (GPCR). The tagged mGluR8a was expressed in rat sympathetic neurons and labelled with fluorescein-conjugated BTX (FL-BTX). The efficacy of FALI was evaluated by monitoring mGluR8a-mediated inhibition of calcium currents (I(Ca)) using whole-cell voltage-clamp techniques. Following either wide-field or laser illumination of FL-BTX-labelled neurons, mGluR8a-mediated I(Ca) inhibition was greatly attenuated whereas holding current and basal I(Ca), measures of non-specific effects, were minimally affected. Sodium azide, a collision quencher of singlet oxygen, reduced the magnitude of FALI-mediated effects supporting a role for reactive oxygen species in the process. Although these results were consistent with an acute inactivation of mGluR8a, the intended target, two findings confounded this interpretation. First, effects on a natively expressed signalling pathway, alpha(2)-adrenergic receptor-mediated I(Ca) modulation, were observed following illumination of neurons expressing FL-BTX-labelled sodium channel beta2 subunits or ionotropic 5-HT(3) receptors, proteins with no overt relationship to GPCR signalling pathways. Second, GPCR-independent I(Ca) modulation induced with intracellular guanylyl imidophosphate was also attenuated by FALI. These data challenge the assumption that the fluorophore-tagged protein is the sole target of FALI and provide evidence that collateral damage to proximal proteins occurs following fluorophore illumination.

Animals↗
PubMed2004

A highly conserved glycine within linker I and the extreme C terminus of G protein alpha subunits interact cooperatively in switching G protein-coupled receptor-to-effector specificity.

Numerous studies have attested to the importance of the extreme C terminus of G protein alpha subunits in determining their selectivity of receptor recognition. We have previously reported that a highly conserved glycine residue within linker I is important for constraining the fidelity of receptor recognition by Galpha(q) proteins. Herein, we explored whether both modules (linker I and extreme C terminus) interact cooperatively in switching G protein-coupled receptor (GPCR)-to-effector specificity and created as models mutant Galpha(q) proteins in which glycine was replaced with various amino acids and the C-terminal five Galpha(q) residues with the corresponding Galpha(i) or Galpha(s) sequence. Coupling properties of the mutated Galpha(q) proteins were determined after coexpression with a panel of 13 G(i)-and G(s) -selective receptors and compared with those of Galpha proteins modified in only one module. Galpha proteins modified in both modules are significantly more efficacious in channeling non-G(q) -selective receptors to G(q)-mediated signaling events compare with those containing each module alone. Additive effects of both modules were observed even if individual modules lacked an effect on GPCR-to-effector specificity. Dually modified Galpha proteins were also superior in conferring high-affinity agonist sites onto a coexpressed GPCR in the absence, but not in the presence, of guanine nucleotides. Together, our data suggest that receptor-G protein coupling selectivity involves cooperative interactions between the extreme C terminus and linker I of Galpha proteins and that distinct determinants of selectivity exist for individual receptors.

Amino Acid Sequence↗
PubMed2005

The origins of diversity and specificity in g protein-coupled receptor signaling.

The modulation of transmembrane signaling by G protein-coupled receptors (GPCRs) constitutes the single most important therapeutic target in medicine. Drugs acting on GPCRs have traditionally been classified as agonists, partial agonists, or antagonists based on a two-state model of receptor function embodied in the ternary complex model. Over the past decade, however, many lines of investigation have shown that GPCR signaling exhibits greater diversity and "texture" than previously appreciated. Signal diversity arises from numerous factors, among which are the ability of receptors to adopt multiple "active" states with different effector-coupling profiles; the formation of receptor dimers that exhibit unique pharmacology, signaling, and trafficking; the dissociation of receptor "activation" from desensitization and internalization; and the discovery that non-G protein effectors mediate some aspects of GPCR signaling. At the same time, clustering of GPCRs with their downstream effectors in membrane microdomains and interactions between receptors and a plethora of multidomain scaffolding proteins and accessory/chaperone molecules confer signal preorganization, efficiency, and specificity. In this context, the concept of agonist-selective trafficking of receptor signaling, which recognizes that a bound ligand may select between a menu of active receptor conformations and induce only a subset of the possible response profile, presents the opportunity to develop drugs that change the quality as well as the quantity of efficacy. As a more comprehensive understanding of the complexity of GPCR signaling is developed, the rational design of ligands possessing increased specific efficacy and attenuated side effects may become the standard mode of drug development.

Animals↗
PubMed2005

Internal PDZ ligands: novel endocytic recycling motifs for G protein-coupled receptors.

Internalization, recycling and lysosomal sorting are key processes that regulate the temporal and spatial signaling of G protein-coupled receptors (GPCRs). Interactions between GPCR intracytosolic sorting signals and adaptor proteins facilitate trafficking through the endocytic pathway. To date only a few sorting signals and molecules that regulate GPCR trafficking have been identified. A study reported in the May 2005 issue of Molecular Pharmacology has now identified an internal PDZ ligand motif that seems to regulate efficient recycling of the ET(A) endothelin receptor. This finding now expands the diversity of GPCR sorting motifs to include internal and C-terminal PDZ ligands, tyrosine-based motifs, and lysine residues capable of being ubiquitinated.

Amino Acid Motifs↗
PubMed2005

Single mutations at Asn295 and Leu305 in the cytoplasmic half of transmembrane alpha-helix domain 7 of the AT1 receptor induce promiscuous agonist specificity for angiotensin II fragments: a pseudo-constitutive activity.

The most striking feature of a G protein-coupled receptor (GPCR) is its highly exclusive agonist specificity. This feature guarantees that a GPCR recognizes only its specific native agonist(s). In this study, we showed that two point mutations of N295S and L305Q enabled the AT(1) receptors to recognize multiple Ang II fragments. Similar to the well established constitutively active AT(1) mutant receptor N111G, the mutations of N295S and L305Q induced an increased production of basal inositol 1,4,5-phosphates in the absence of exogenous Ang II when expressed in HEK293 cells. Distinct from the N111G, however, is the fact that the increased basal activity disappeared in COS-7 cells because of the lack of endogenous Ang II fragments produced by the cells-a pseudo-constitutive activity. It is surprising that the Ang II analog [Sar(1),Ile(4),Ile(8)]Ang II and the native angiotensin II fragments Ang 1-7, Ang IV, and Ang 5-8, which are inactive in activating the wild-type receptor, activated N295S and L305Q. Results generated by lowering the Na(+) concentration suggest that the mutant N295S and L305Q may be trapped in neutral conformational states (R(N)). These data allow us to identify for the first time a novel pattern of GPCR mutations with a broad spectrum of agonist specificity, suggesting possible existence of functional GPCRs in nature that are activated through conformational "selection" rather than "induction" mechanisms.

Angiotensin II↗
PubMed2006

Class B GPCRs: a hidden agonist within?

Class B G protein-coupled receptors (GPCRs) regulate a wide range of endocrine and neuroendocrine functions and are endogenously stimulated by moderately large peptide hormones. Current evidence suggests that the carboxyl termini of cognate peptides bind to the amino terminus of their G protein-coupled receptors (GPCRs) and that the peptides' amino terminal segments then dock to the heptahelical receptor portion to induce signaling. In this issue of Molecular Pharmacology, Dong et al. (p. 206) propose an alternative model of ligand-induced class B GPCR activation. Based primarily on studies with the secretin receptor, a prototype class B family member, they provide evidence that the endogenous peptide hormone does not function as an activator per se. Instead, this hormone (secretin) exposes a hidden, built-in agonist epitope that is present within the amino terminus of its target GPCR. Isolated oligopeptide fragments containing this epitope act as full agonists on the secretin receptor despite their lack of amino acid homology with the secretin hormone. These nonconventional agonists can be minimized to tripeptide molecules and still maintain biological activity. The study to be discussed introduces a novel paradigm of class B GPCR function, and may facilitate the elusive goal of finding small molecule agonist drugs for this therapeutically attractive group of receptors.

Animals↗