A database of mutants and effects of site-directed mutagenesis experiments on G protein-coupled receptors

A database system and computer programs for storage and retrieval of information about guanine nucleotide-binding protein (G protein) -coupled receptor mutants and associated biological effects have been developed. Mutation data on the receptors were collected from the literature and a database of mutants and effects of mutations was developed. The G protein-coupled receptor, family A, point mutation database (GRAP) provides detailed information on ligand-binding and signal transduction properties of more than 2130 receptor mutants. The amino acid sequences of receptors for which mutation experiments have been reported were aligned, and from this alignment mutation data may be retrieved. Alternatively, a search form allowing detailed specification of which mutants to retrieve may be used, for example, to search for specific amino acid substitutions, substitutions in specific protein domains or reported biological effects. Furthermore, ligand and bibliographic oriented queries may be performed. GRAP is available on the Internet (URL: http://www-grap.fagmed.uit.no/GRAP/homepage.html ) using the World-Wide Web system. © 1996 Wiley-Liss, Inc.     

NRMD: Nuclear Receptor Mutation Database

The NRMD is a database for nuclear receptor mutation information. It includes mutation information from SWISS-PROT/TrEMBL, several web-based mutation data resources, and data extracted from the literature in a fully automatic manner. Because it is also possible to add mutations manually, a hundred mutations were added for completeness. At present, the NRMD contains information about 893 mutations in 54 nuclear receptors. A common numbering scheme for all nuclear receptors eases the use of the information for many kinds of studies. The NRMD is freely available to academia and industry as a stand-alone version at: www.receptors.org/NR/.     

Automatic extraction of protein point mutations using a graph bigram association

Protein point mutations are an essential component of the evolutionary and experimental analysis of protein structure and function. While many manually curated databases attempt to index point mutations, most experimentally generated point mutations and the biological impacts of the changes are described in the peer-reviewed published literature. We describe an application, Mutation GraB (Graph Bigram), that identifies, extracts, and verifies point mutations from biomedical literature. The principal problem of point mutation extraction is to link the point mutation with its associated protein and organism of origin. Our algorithm uses a graph-based bigram traversal to identify these relevant associations and exploits the Swiss-Prot protein database to verify this information. The graph bigram method is different from other models for point mutation extraction in that it incorporates frequency and positional data of all terms in an article to drive the point mutation–protein association. Our method was tested on 589 articles describing point mutations from the G protein–coupled receptor (GPCR), tyrosine kinase, and ion channel protein families. We evaluated our graph bigram metric against a word-proximity metric for term association on datasets of full-text literature in these three different protein families. Our testing shows that the graph bigram metric achieves a higher F-measure for the GPCRs (0.79 versus 0.76), protein tyrosine kinases (0.72 versus 0.69), and ion channel transporters (0.76 versus 0.74). Importantly, in situations where more than one protein can be assigned to a point mutation and disambiguation is required, the graph bigram metric achieves a precision of 0.84 compared with the word distance metric precision of 0.73. We believe the graph bigram search metric to be a significant improvement over previous search metrics for point mutation extraction and to be applicable to text-mining application requiring the association of words.     

GPCRDB information system for G protein-coupled receptors

The GPCRDB is a molecular class-specific information system that collects, combines, validates and disseminates heterogeneous data on G protein-coupled receptors (GPCRs). The database stores data on sequences, ligand binding constants and mutations. The system also provides computationally derived data such as sequence alignments, homology models, and a series of query and visualization tools. The GPCRDB is updated automatically once every 4-5 months and is freely accessible at http://www.gpcr.org/7tm/.     

The Amino Terminus with a Conserved Glutamic Acid of G Protein-Coupled Receptor Kinases Is Indispensable for Their Ability to Phosphorylate Photoactivated Rhodopsin

To investigate functions of the consensus amino terminus of G protein-coupled receptor kinases (GRKs), two amino terminus-truncated mutants (delta30 or delta15) and two single-amino-acid mutants of conserved acidic residues (D2A or E7A) of human GRK1 were constructed and expressed in human embryonic kidney 293 cells. It was shown that truncated mutations and one single-point mutation (E7A) greatly decreased GRK1's activity to phosphorylate photoactivated rhodopsin (Rho*), whereas the abilities of these mutants to phosphorylate a synthetic peptide substrate and to translocate from cytosol to rod outer segments on light activation were unaffected. Further experiments demonstrated that the same truncated mutations (delta30 or delta15) of GRK2, representative of another GRK subfamily, also abolished the kinase's activity toward Rho*. The similar single-point mutation (E5A) of GRK2 heavily impaired its phosphorylation of Rho* but did not alter its ability to phosphorylate the peptide, and the G329-rhodopsin-augmented peptide phosphorylation by GRK2 (E5A) remained unchanged. Our data, taken together, suggest that the amino terminus as well as a conserved glutamic acid in the region of GRKs appears essential for their ability to functionally interact with G protein-coupled receptors.     

Automated extraction of mutation data from the literature: application of MuteXt to G protein-coupled receptors and nuclear hormone receptors

MOTIVATION: The amount of genomic and proteomic data that is published daily in the scientific literature is outstripping the ability of experimental scientists to stay current. Reviews, the traditional medium for collating published observations, are also unable to keep pace. For some specific classes of information (e.g. sequences and protein structures), obligatory data deposition policies have helped. However, a great deal of other valuable information is spread throughout the literature hindering coherent access. We are involved in the Molecular Class-Specific Information System (MCSIS) project, a collaborative effort to design and automate the maintenance of protein family databases. The first two databases, the GPCRDB and NucleaRDB, are focused on G protein-coupled receptors (GPCRs) and nuclear hormone receptors (NRs), respectively. The main aim of the MCSIS project is to gather heterogeneous data from across a variety of electronic and literature sources in order to draw new inferences about the target protein families. RESULTS: We present a computational method that identifies and extracts mutation data from the scientific literature. We focused on the extraction of single point mutations for the GPCR and NR superfamilies. After validation by plausibility filters, the mutation data is integrated into the corresponding MCSIS where it is combined with structural and sequence information already stored in these databases. We extracted and validated 2736 true point mutations from 914 articles on GPCRs and 785 true point mutations from 1094 articles on NRs. The current version of our automated extraction algorithm identifies 49.3% of the GPCR point mutations with a specificity of 87.9%, and 64.5% of the NR point mutations with a specificity of 85.8%. MuteXt routinely analyzes 100 electronic articles in approximately 1 h.     

Mutagenesis and peptide analysis of the DRY motif in the alpha2A adrenergic receptor: evidence for alternate mechanisms in G protein-coupled receptors

In G protein-coupled receptors (GPCRs), a conserved aspartic acid in the DRY motif at the cytoplasmic end of helix 3 regulates the transition to the active state, while the adjacent arginine is crucial for G protein activation. To examine the functions of these two residues, we made D130I and R131Q mutations in the alpha2A adrenergic receptor (AR). We demonstrate that, unlike other GPCRs, the alpha2A AR is not constitutively activated by the D130I mutation, although the mutation increases agonist affinity. While the R131Q mutation severely disrupts function, it decreases rather than increasing agonist affinity as seen in other GPCRs. We then investigated the molecular effects of the same mutations in a peptide model and showed that Arg131 is not required for peptide-mediated G protein activation. These results indicate that the alpha2A AR does not follow the conventional GPCR mechanistic paradigm with respect to the function of the DRY motif.     

Structure and function of the human calcium-sensing receptor: insights from natural and engineered mutations and allosteric modulators

The human extracellular Ca(2+)-sensing receptor (CaR), a member of the G protein-coupled receptor family 3, plays a key role in the regulation of extracellular calcium homeostasis. It is one of just a few G protein-coupled receptors with a large number of naturally occurring mutations identified in patients. In contrast to the small sizes of its agonists, this large dimeric receptor consists of domains with topologically distinctive orthosteric and allosteric sites. Information derived from studies of naturally occurring mutations, engineered mutations, allosteric modulators and crystal structures of the agonist-binding domain of homologous type 1 metabotropic glutamate receptor and G protein-coupled rhodopsin offers new insights into the structure and function of the CaR.     

Mutation of Three Residues in the Third Intracellular Loop of the Dopamine D2 Receptor Creates an Internalization-defective Receptor

Arrestins mediate desensitization and internalization of G protein-coupled receptors and also direct receptor signaling toward heterotrimeric G protein-independent signaling pathways. We previously identified a four-residue segment (residues 212–215) of the dopamine D2 receptor that is necessary for arrestin binding in an in vitro heterologous expression system but that also impairs receptor expression. We now describe the characterization of additional mutations at that arrestin binding site in the third intracellular loop. Mutating two (residues 214 and 215) or three (residues 213–215) of the four residues to alanine partially decreased agonist-induced recruitment of arrestin3 without altering activation of a G protein. Arrestin-dependent receptor internalization, which requires arrestin binding to β2-adaptin (the β2 subunit of the clathrin-associated adaptor protein AP2) and clathrin, was disproportionately affected by the three-residue mutation, with no agonist-induced internalization observed even in the presence of overexpressed arrestin or G protein-coupled receptor kinase 2. The disjunction between arrestin recruitment and internalization could not be explained by alterations in the time course of the receptor-arrestin interaction, the recruitment of G protein-coupled receptor kinase 2, or the receptor-induced interaction between arrestin and β2-adaptin, suggesting that the mutation impairs a property of the internalization complex that has not yet been identified.     

Chemical genetic engineering of G protein-coupled receptor kinase 2

G protein-coupled receptor kinases (GRKs) play a pivotal role in receptor regulation. Efforts to study the acute effects of GRKs in intact cells have been limited by a lack of specific inhibitors. In the present study we have developed an engineered version of GRK2 that is specifically and reversibly inhibited by the substituted nucleotide analog 1-naphthyl-PP1 (1Na-PP1), and we explored GRK2 function in regulated internalization of the mu-opioid receptor (muOR). A previously described method that conferred analog sensitivity on various kinases, by introducing a space-creating mutation in the conserved active site, failed when applied to GRK2 because the corresponding mutation (L271G) rendered the mutant kinase (GRK2-as1) catalytically inactive. A sequence homology-based approach was used to design second-site suppressor mutations. A C221V second-site mutation produced a mutant kinase (GRK2-as5) with full functional activity and analog sensitivity as compared with wild-type GRK2 in vitro and in intact cells. The role of GRK2-as5 activity in the membrane trafficking of the muOR was also characterized. Morphine-induced internalization was completely blocked when GRK2-as5 activity was inhibited before morphine application. However, inhibition of GRK2-as5 during recycling and reinternalization of the muOR did not attenuate these processes. These results suggest there is a difference in the GRK requirement for initial ligand-induced internalization of a G protein-coupled receptor compared with subsequent rounds of reinternalization.     

Molecular modeling of human MT2 melatonin receptor: the role of Val204, Leu272 and Tyr298 in ligand binding

A model of the helical part of the human MT2 melatonin (hMT2) receptor, a member of the G protein-coupled receptors superfamily has been generated, based on the structure of bovine rhodopsin. Modeling has been combined with site-directed mutagenesis to investigate the role of the specific amino acid residues within the transmembrane domains (TM) numbers V, VI and VII of hMT2 receptor in the interaction with 2-iodomelatonin. Saturation binding assays with 2-iodomelatonin demonstrated that the substitution V204A (TMV) resulted in total loss of binding while the mutation V205A had no effect. The replacement of F209 with alanine led to a significant decrease in the Bmax value of receptor binding while mutations V205A and F209A also within TM V did not significantly change binding properties of the hMT2 receptor. In the case of TM VI, the substitution G271T caused substantial decrease in 2-iodomelatonin binding to the hMT2 receptor. The change L272A (TM VI) as well as mutation Y298A within TM VII completely abolished ligand binding to the receptor. These data suggest that several new amino acid residues within TM V, VI and VII are involved in ligand-MT2 receptor interaction.     

Random mutagenesis of the M3 muscarinic acetylcholine receptor expressed in yeast. Identification of point mutations that "silence" a constitutively active mutant M3 receptor and greatly impair receptor/G protein coupling

The M3 muscarinic receptor is a prototypical member of the class I family of G protein-coupled receptors (GPCRs). To facilitate studies on the structural mechanisms governing M3 receptor activation, we generated an M3 receptor-expressing yeast strain (Saccharomyces cerevisiae) that requires agonist-dependent M3 receptor activation for cell growth. By using receptor random mutagenesis followed by a genetic screen in yeast, we initially identified a point mutation at the cytoplasmic end of transmembrane domain (TM) VI (Q490L) that led to robust agonist-independent M3 receptor signaling in both yeast and mammalian cells. To explore further the molecular mechanisms by which point mutations can render GPCRs constitutively active, we subjected a region of the Q490L mutant M3 receptor that included TM V-VII to random mutagenesis. We then applied a yeast genetic screen to identify second-site mutations that could suppress the activating effects of the Q490L mutation and restore wild-type receptor-like function to the Q490L mutant receptor. This analysis led to the identification of 12 point mutations that allowed the Q490L mutant receptor to function in a fashion similar to the wild-type receptor. These amino acid substitutions mapped to two distinct regions of the M3 receptor, the exofacial segments of TM V and VI and the cytoplasmic ends of TM V-VII. Strikingly, in the absence of the activating Q490L mutation, all recovered point mutations severely reduced the efficiency of receptor/G protein coupling, indicating that the targeted residues play important roles in receptor activation and/or receptor/G protein coupling. This strategy should be generally applicable to identify sites in GPCRs that are critically involved in receptor function.     

A conserved Asn in transmembrane helix 7 is an on/off switch in the activation of the thyrotropin receptor

The thyrotropin (TSH) receptor is an interesting model to study G protein-coupled receptor activation as many point mutations can significantly increase its basal activity. Here, we identified a molecular interaction between Asp(633) in transmembrane helix 6 (TM6) and Asn(674) in TM7 of the TSHr that is crucial to maintain the inactive state through conformational constraint of the Asn. We show that these residues are perfectly conserved in the glycohormone receptor family, except in one case, where they are exchanged, suggesting a direct interaction. Molecular modeling of the TSHr, based on the high resolution structure of rhodopsin, strongly favors this hypothesis. Our approach combining site-directed mutagenesis with molecular modeling shows that mutations disrupting this interaction, like the D633A mutation in TM6, lead to high constitutive activation. The strongly activating N674D (TM7) mutation, which in our modeling breaks the TM6-TM7 link, is reverted to wild type-like behavior by an additional D633N mutation (TM6), which would restore this link. Moreover, we show that the Asn of TM7 (conserved in most G protein-coupled receptors) is mandatory for ligand-induced cAMP accumulation, suggesting an active role of this residue in activation. In the TSHr, the conformation of this Asn residue of TM7 would be constrained, in the inactive state, by its Asp partner in TM6.     

Functional rescue of the nephrogenic diabetes insipidus-causing vasopressin V2 receptor mutants G185C and R202C by a second site suppressor mutation

Mutations in the gene of the G protein-coupled vasopressin V2 receptor (V2 receptor) cause X-linked nephrogenic diabetes insipidus (NDI). Most of the missense mutations on the extracellular face of the receptor introduce additional cysteine residues. Several groups have proposed that these residues might disrupt the conserved disulfide bond of the V2 receptor. To test this hypothesis, we first calculated a structure model of the extracellular receptor domains. The model suggests that the additional cysteine residues may form a second disulfide bond with the free, nonconserved extracellular cysteine residue Cys-195 rather than impairing the conserved bond. To address this question experimentally, we used the NDI-causing mutant receptors G185C and R202C. Their Cys-195 residues were replaced by alanine to eliminate the hypothetical second disulfide bonds. This second site mutation led to functional rescue of both NDI-causing mutant receptors, strongly suggesting that the second disulfide bonds are indeed formed. Furthermore we show that residue Cys-195, which is sensitive to "additional cysteine" mutations, is not conserved among the V2 receptors of other species and that the presence of an uneven number of extracellular cysteine residues, as in the human V2 receptor, is rare among class I G protein-coupled receptors.     

Experimental evidence for lack of homodimerization of the G protein-coupled human N-formyl peptide receptor

A large number of G protein-coupled receptors have been shown to form homodimers based on a number of different techniques such as receptor coimmunoprecipitation, cross-linking, and fluorescence resonance energy transfer. In addition, functional assays of cells coexpressing a mutant receptor with a wild-type receptor have shown receptor phenotypes that can best be explained through dimerization. We asked whether the human neutrophil N-formyl peptide receptor (FPR) forms dimers in Chinese hamster ovary cells by coexpressing wild-type FPR with one of two mutants: D71A, which is uncoupled from G protein, and N297A, which has a defect in receptor phosphorylation and endocytosis. Experiments measuring chemotaxis, ligand-induced release of intracellular calcium, and p42/44 mitogen-activated protein kinase activation did not show an inhibitory effect of the coexpressed FPR D71A mutant. Coexpressed wild-type receptor was efficiently internalized, but failed to correct the endocytosis defects of the D71A and the N297A mutants. To explore the possibility that the mutations themselves prevented dimerization, we examined the coimmunoprecipitation of differentially epitope-tagged FPR. Immunoprecipitation of hemagglutinin-tagged FPR failed to coimmunoprecipitate coexpressed c-myc-tagged FPR and vice versa. Together, these data suggest that, unlike many other G protein-coupled receptors, FPR does not form homodimers.     

Constitutive activation of the opioid receptor-like (ORL1) receptor by mutation of Asn133 to tryptophan in the third transmembrane region

We have introduced a series of point mutations into the human opioid receptor-like (ORL1) receptor and characterized them for their ability to constitutively activate G protein-coupled receptor signalling pathways. Among the 12 mutants generated, mutation at Asn133 (N133W) gave increased basal signalling through three separate pathways. N133W increased the basal activity of G14- and G16-dependent pathways by two- to three-fold. The constitutive activity of the mutant was confirmed by the finding that the enhanced activity is dependent on the level of receptor expression. In HEK-293 cells stably expressing N133W, signalling through Gi/o-dependent pathways was also observed. Radioligand binding studies revealed that the affinity for nociceptin of the wild-type ORL1 receptor and the N133W mutant do not differ significantly, suggesting that the ligand binding and signalling functions of constitutively active mutants of G protein-coupled receptors are not necessarily intrinsically linked. In conclusion, our results demonstrate that a mutation in the third transmembrane domain is able to increase the basal signalling activity of the human ORL1 receptor.     

Random mutagenesis of the M3 muscarinic acetylcholine receptor expressed in yeast: identification of second-site mutations that restore function to a coupling-deficient mutant M3 receptor

The M(3) muscarinic receptor is a prototypical member of the class A family of G protein-coupled receptors (GPCRs). To gain insight into the structural mechanisms governing agonist-mediated M(3) receptor activation, we recently developed a genetically modified yeast strain (Saccharomyces cerevisiae) which allows the efficient screening of large libraries of mutant M(3) receptors to identify mutant receptors with altered/novel functional properties. Class A GPCRs contain a highly conserved Asp residue located in transmembrane domain II (TM II; corresponding to Asp-113 in the rat M(3) muscarinic receptor) which is of fundamental importance for receptor activation. As observed previously with other GPCRs analyzed in mammalian expression systems, the D113N point mutation abolished agonist-induced receptor/G protein coupling in yeast. We then subjected the D113N mutant M(3) receptor to PCR-based random mutagenesis followed by a yeast genetic screen to recover point mutations that can restore G protein coupling to the D113N mutant receptor. A large scale screening effort led to the identification of three such second-site suppressor mutations, R165W, R165M, and Y250D. When expressed in the wild-type receptor background, these three point mutations did not lead to an increase in basal activity and reduced the efficiency of receptor/G protein coupling. Similar results were obtained when the various mutant receptors were expressed and analyzed in transfected mammalian cells (COS-7 cells). Interestingly, like Asp-113, Arg-165 and Tyr-250, which are located at the cytoplasmic ends of TM III and TM V, respectively, are also highly conserved among class A GPCRs. Our data suggest a conformational link between the highly conserved Asp-113, Arg-165, and Tyr-250 residues which is critical for receptor activation.     

APC gene: database of germline and somatic mutations in human tumors and cell lines.

A database (http://perso.curie.fr/tsoussi ) is described, in which over 1000 mutations in the human APC gene of tumors (colon cancer predominantly) are compiled from the literature. It includes both molecular information about the mutations and clinical data about the patients. Software has been designed to analyse all this information in the database.     

Paired cysteine mutagenesis to establish the pattern of disulfide bonds in the functional intact secretin receptor

The amino-terminal domain of class B G protein-coupled receptors contains six conserved cysteine residues involved in structurally and functionally critical disulfide bonds. The mapping of these bonds has been unclear, with one pattern based on biochemical and NMR structural characterizations of refolded, nonglycosylated amino-terminal fragments, and another pattern derived from functional characterizations of intact receptors having paired cysteine mutations. In the present study, we determined the disulfide bonding pattern of the prototypic class B secretin receptor by applying the same paired cysteine mutagenesis approach and confirming the predicted bonding pattern with proteolytic cleavage of intact functional receptor. As expected, systematic mutation to serine of the six conserved cysteine residues within this region of the secretin receptor singly and in pairs resulted in loss of function of most constructs. Notable exceptions were single mutations of the 4th and 6th cysteine residues and paired mutations involving the 1st and 3rd, 2nd and 5th, and 4th and 6th conserved cysteines, with secretin eliciting statistically significant cAMP responses above basal levels of activation for each of these constructs. Immunofluorescence microscopy confirmed similar levels of plasma membrane expression for each of the mutated receptors. Furthermore, cyanogen bromide cleaved a series of wild type and mutant secretin receptors, yielding patterns that agreed with our paired cysteine mutagenesis results. In conclusion, these data suggest the same pattern of disulfide bonding as that predicted previously by NMR and thus support a consistent pattern of amino-terminal disulfide bonds in class B G protein-coupled receptors.     

The Relaxin Receptor (RXFP1) Utilizes Hydrophobic Moieties on a Signaling Surface of Its N-terminal Low Density Lipoprotein Class A Module to Mediate Receptor Activation

The peptide hormone relaxin is showing potential as a treatment for acute heart failure. Although it is known that relaxin mediates its actions through the G protein-coupled receptor relaxin family peptide receptor 1 (RXFP1), little is known about the molecular mechanisms by which relaxin binding results in receptor activation. Previous studies have highlighted that the unique N-terminal low density lipoprotein class A (LDLa) module of RXFP1 is essential for receptor activation, and it has been hypothesized that this module is the true “ligand” of the receptor that directs the conformational changes necessary for G protein coupling. In this study, we confirmed that an RXFP1 receptor lacking the LDLa module binds ligand normally but cannot signal through any characterized G protein-coupled receptor signaling pathway. Furthermore, we comprehensively examined the contributions of amino acids in the LDLa module to RXFP1 activity using both gain-of-function and loss-of-function mutational analysis together with NMR structural analysis of recombinant LDLa modules. Gain-of-function studies with an inactive RXFP1 chimera containing the LDLa module of the human LDL receptor (LB2) demonstrated two key N-terminal regions of the module that were able to rescue receptor signaling. Loss-of-function mutations of residues in these regions demonstrated that Leu-7, Tyr-9, and Lys-17 all contributed to the ability of the LDLa module to drive receptor activation, and judicious amino acid substitutions suggested this involves hydrophobic interactions. Our results demonstrate that these key residues contribute to interactions driving the active receptor conformation, providing further evidence of a unique mode of G protein-coupled receptor activation.