RESUMEN
Recently three orphan G-protein coupled receptors, RAIG1, GPRC5B and GPRC5C, with homology to members of family C (metabotropic glutamate receptor-like) have been identified. Using the protein sequences of these receptors as queries we identified overlapping expressed sequence tags which were predicted to encode an additional subtype. The full length coding regions of mouse mGprc5d and human GPRC5D were cloned and shown to contain predicted open reading frames of 300 and 345 amino acids, respectively. GPRC5D has seven putative transmembrane segments and is expressed in the cell membrane. The four human receptor subtypes, which we assign to group 5 of family C GPCRs, show 31-42% amino acid sequence identity to each other and 20-25% sequence identity to the transmembrane domains of metabotropic glutamate receptor subtypes 2 and 3 and other family C members. In contrast to the remaining family C members, the group 5 receptors have short amino terminal domains of some 30-50 amino acids. GPRC5D was shown to be clustered with RAIG1 on chromosome 12p13.3 and like RAIG1 and GPRC5B to consist of three exons, the first exon being the largest containing all seven transmembrane segments. GPRC5D mRNA is widely expressed in the peripheral system but all four receptors show distinct expression patterns. Interestingly, mRNA levels of all four group 5 receptors were found in medium to high levels in the kidney, pancreas and prostate and in low to medium levels in the colon and the small intestine, whereas other organs only express a subset of the genes. In an attempt to delineate the signal transduction pathway(s) of the orphan receptors, a series of chimeric receptors containing the amino terminal domain of the calcium sensing receptor or metabotropic glutamate receptor subtype 1, and the seven transmembrane domain of the orphan receptors were constructed and tested in binding and functional assays.
Asunto(s)
Proteínas de Unión al GTP/metabolismo , Receptores de Superficie Celular/genética , Receptores Acoplados a Proteínas G , Receptores de Glutamato Metabotrópico/genética , Proteínas Recombinantes de Fusión/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Membrana Celular/metabolismo , Clonación Molecular , Biblioteca de Genes , Proteínas Fluorescentes Verdes , Humanos , Riñón/metabolismo , Proteínas Luminiscentes/química , Proteínas Luminiscentes/genética , Masculino , Ratones , Datos de Secuencia Molecular , Páncreas/metabolismo , Próstata/metabolismo , Ácido Quiscuálico , Receptores de Superficie Celular/química , Receptores de Glutamato Metabotrópico/química , Proteínas Recombinantes de Fusión/química , Alineación de Secuencia , Homología de Secuencia , Transducción de Señal , Transfección , Tretinoina , Células Tumorales CultivadasRESUMEN
We have characterized platelet-derived growth factor (PDGF) C, a novel growth factor belonging to the PDGF family. PDGF-C is a multidomain protein with the N-terminal region homologous to the extracellular CUB domain of neuropilin-1, and the C-terminal region consists of a growth factor domain (GFD) with homology to vascular endothelial growth factor (25%) and PDGF A-chain (23%). A serum-sensitive cleavage site between the two domains allows release of the GFD from the CUB domain. Competition binding and immunoprecipitation studies on cells bearing both PDGF alpha and beta receptors reveal a high affinity binding of recombinant GFD (PDGF-CC) to PDGF receptor-alpha homodimers and PDGF receptor-alpha/beta heterodimers. PDGF-CC exhibits greater mitogenic potency than PDGF-AA and comparable or greater mitogenic activity than PDGF-AB and PDGF-BB on several mesenchymal cell types. Analysis of PDGF-CC in vivo in a diabetic mouse model of delayed wound healing showed that PDGF-CC significantly enhanced repair of a full-thickness skin excision. Together, these studies describe a third member of the PDGF family (PDGF-C) as a potent mitogen for cells of mesenchymal origin in in vitro and in vivo systems with a binding pattern similar to PDGF-AB.
Asunto(s)
Factor de Crecimiento Derivado de Plaquetas/metabolismo , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Secuencia de Aminoácidos , Animales , Aorta/crecimiento & desarrollo , Línea Celular , Cricetinae , ADN Complementario , Diabetes Mellitus Experimental/fisiopatología , Humanos , Linfocinas , Ratones , Datos de Secuencia Molecular , Factor de Crecimiento Derivado de Plaquetas/química , Factor de Crecimiento Derivado de Plaquetas/genética , Factor de Crecimiento Derivado de Plaquetas/fisiología , Unión Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Timidina/metabolismo , Cicatrización de Heridas/fisiologíaRESUMEN
The metabotropic glutamate receptors (mGluRs) belong to family C of the G-protein-coupled receptor (GPCR) superfamily. The receptors are characterized by having unusually long amino-terminal domains (ATDs), to which agonist binding has been shown to take place. Previously, we have constructed a molecular model of the ATD of mGluR1 based on a weak amino acid sequence similarity with a bacterial periplasmic binding protein. The ATD consists of two globular lobes, which are speculated to contract from an "open" to a "closed" conformation following agonist binding. In the present study, we have created a Zn(2+) binding site in mGluR1b by mutating the residue Lys(260) to a histidine. Zinc acts as a noncompetitive antagonist of agonist-induced IP accumulation on the K260H mutant with an IC(50) value of 2 microm. Alanine mutations of three potential "zinc coligands" in proximity to the introduced histidine in K260H knock out the ability of Zn(2+) to antagonize the agonist-induced response. Zn(2+) binding to K260H does not appear to affect the dimerization of the receptor. Instead, we propose that binding of zinc has introduced a structural constraint in the ATD lobe, preventing the formation of a "closed" conformation, and thus stabilizing a more or less inactive "open" form of the ATD. This study presents the first metal ion site constructed in a family C GPCR. Furthermore, it is the first time a metal ion site has been created in a region outside of the seven transmembrane regions of a GPCR and the loops connecting these. The findings offer valuable insight into the mechanism of ATD closure and family C receptor activation. Furthermore, the findings demonstrate that ATD regions other than those participating in agonist binding could be potential targets for new generations of ligands for this family of receptors.
Asunto(s)
Receptores de Glutamato Metabotrópico/química , Receptores de Glutamato Metabotrópico/metabolismo , Zinc/metabolismo , Alanina/química , Animales , Sitios de Unión , Western Blotting , Células CHO , Línea Celular , Membrana Celular/metabolismo , Cloruros/farmacología , Cricetinae , Relación Dosis-Respuesta a Droga , Ácido Glutámico/metabolismo , Histidina/química , Humanos , Immunoblotting , Concentración 50 Inhibidora , Fosfatos de Inositol/metabolismo , Iones , Cinética , Ligandos , Lisina/química , Modelos Moleculares , Mutación , Plásmidos/metabolismo , Mutación Puntual , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , Transfección , Zinc/química , Compuestos de Zinc/farmacologíaRESUMEN
The calcium-sensing receptor (CaR) belongs to family C of the G-protein-coupled receptor superfamily. To date 14 activating mutations in CaR showing increased sensitivity to Ca(2+) have been identified in humans with autosomal dominant hypocalcemia. Four of these activating mutations are found in the Ala(116)-Pro(136) region of CaR, indicating that this part of the receptor is particularly sensitive to mutation-induced activation. This region was subjected to random saturation mutagenesis, and 219 mutant receptor clones were isolated and screened pharmacologically in a high throughput screening assay. Selected mutants were characterized further in an inositol phosphate assay. The vast majority of the mutants tested displayed an increased affinity for Ca(2+). Furthermore, 21 of the mutants showed increased basal activity in the absence of agonist. This constitutive activity was not diminished when the mutations were transferred to a chimeric receptor Ca/1a consisting of the amino-terminal domain of the CaR and the 7 transmembrane and intracellular domains of the metabotropic glutamate receptor mGluR1a. CPCCOEt, a noncompetitive antagonist acting at the 7 transmembrane domain of mGluR1a, suppressed the elevated basal response of the constitutively activated Ca/1a mutants demonstrating inverse agonist activity of CPCCOEt. Taken together, our results demonstrate that the Ala(116)-Pro(136) region is of key importance for the maintenance of the inactive conformation of CaR.
Asunto(s)
Receptores de Superficie Celular/química , Receptores de Superficie Celular/metabolismo , Células 3T3 , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas de Unión al GTP/metabolismo , Ratones , Datos de Secuencia Molecular , Mutagénesis , Conformación Proteica , Receptores Sensibles al Calcio , Receptores de Superficie Celular/genética , Transducción de Señal , Relación Estructura-ActividadRESUMEN
The metabotropic glutamate receptors belong to family C of the G-protein coupled receptor superfamily. These receptors all possess large extracellular amino terminal domains, where agonist binding takes place. We have previously constructed a molecular model of the amino terminal domain of the mGlu(1) receptor based on a weak amino acid sequence similarity with a family of bacterial periplasmic binding proteins (PBPs). The residues Ser(165) and Thr(188) were demonstrated to be involved in agonist binding to the receptor. Here, we report that mutation of Arg(78) in the mGlu(1b) receptor to leucine or glutamate completely knocks out [3H]quisqualic acid binding to the receptor. The constructed mutants, R78L and R78E, have also been characterized in a inositol phosphate assay. Here, the potency of (S)-glutamic acid and (S)-quisqualic acid was reduced 1000- and 100-fold, respectively, on R78L compared to the wild type (WT) receptor. (S)-Quisqualic acid was as potent on mutant R78E as it was on R78L, whereas (S)-glutamic acid was unable to activate R78E significantly at concentrations up to 10 mM. In conclusion, Arg(78) appears to be essential for agonist binding to the mGlu(1) receptor, most likely, through the formation of an ionic bond between its positively charged side chain and the distal acid group of the agonists. Furthermore, the different impact of the two mutations on (S)-glutamic acid and (S)-quisqualic acid potencies strongly indicates that while Arg(78) appears to be a common site of interaction for the agonists, the Group I subtype selectivity of (S)-quisqualic acid is probably determined by other residues in the amino terminal domain.
Asunto(s)
Sitios de Unión/fisiología , Agonistas de Aminoácidos Excitadores/farmacología , Receptores de Glutamato Metabotrópico/agonistas , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Arginina/fisiología , Sitios de Unión/genética , Unión Competitiva , Línea Celular , Relación Dosis-Respuesta a Droga , Ácido Glutámico/farmacología , Humanos , Fosfatos de Inositol/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Estructura Terciaria de Proteína , Ácido Quiscuálico/farmacología , Receptores de Glutamato Metabotrópico/química , Receptores de Glutamato Metabotrópico/genética , Homología de Secuencia de AminoácidoRESUMEN
The calcium-sensing receptor (CaR) is a G-protein-coupled receptor that displays 19-25% sequence identity to the gamma-aminobutyric acid type B (GABAB) and metabotropic glutamate (mGlu) receptors. All three groups of receptors have a large amino-terminal domain (ATD), which for the mGlu receptors has been shown to bind the endogenous agonist. To investigate whether the agonist-binding domain of the CaR also is located in the ATD, we constructed a chimeric receptor named Ca/1a consisting of the ATD of CaR and the seven transmembrane region and C terminus of mGlu1a. The Ca/1a receptor stimulated inositol phosphate production when exposed to the cationic agonists Ca2+, Mg2+, and Ba2+ in transiently transfected tsA cells (a transformed HEK 293 cell line). The pharmacological profile of Ca/1a (EC50 values of 3.3, 2.6, and 3.9 mM for these cations, respectively) was very similar to that of the wild-type CaR (EC50 values of 3.2, 4.7, and 4.1 mM, respectively). For the mGlu1a receptor, it has been shown that Ser-165 and Thr-188, which are located in the ATD, are involved in the agonist binding. An alignment of CaR with the mGlu receptors showed that these two amino acid residues have been conserved in CaR as Ser-147 and Ser-170, respectively. Each of these residues was mutated to alanines and tested pharmacologically using the endogenous agonist Ca2+. CaR-S147A showed an impaired function as compared with wild-type CaR both with respect to potency of Ca2+ (4-fold increase in EC50) and maximal response (79% of wild-type response). CaR-S170A showed no significant response to Ca2+ even at 50 mM concentration. In contrast, each of the two adjacent mutations, S169A and S171A, resulted in pharmacological profiles almost identical to that of the wild-type receptor. These data demonstrate that Ser-170 and to some extent Ser-147 are involved in the Ca2+ activation of the CaR, and taken together, our results reveal a close resemblance of the activation mechanism between the CaR and the mGlu receptors.
Asunto(s)
Calcio/metabolismo , Receptores de Superficie Celular/metabolismo , Secuencia de Aminoácidos , Animales , Células Cultivadas , Relación Dosis-Respuesta a Droga , Humanos , Datos de Secuencia Molecular , Mutación Puntual , Ratas , Receptores Sensibles al Calcio , Receptores de Superficie Celular/genética , Receptores de Glutamato Metabotrópico/metabolismo , Alineación de Secuencia , Relación Estructura-ActividadRESUMEN
By exchanging portions of the AMPA receptor subunit GluR3 and the kainate receptor subunit GluR6, we have identified two discontinuous segments of approximately 150 amino acid residues each that control the agonist pharmacology of these glutamate receptors. The first segment (S1) is adjacent and N-terminal to the putative transmembrane domain 1 (TM1), whereas the second segment (S2) is located between the putative TM3 and TM4. Only the simultaneous exchange of S1 and S2 converts the pharmacological profile of the recipient to that of the donor subunit. The two segments identified in this study share sequence similarities with the ligand-binding site of several bacterial periplasmic amino acid-binding proteins. Based on the X-ray structure of these proteins, we propose a model for the glutamate-binding site of ionotropic glutamate receptors.
Asunto(s)
Proteínas Bacterianas , Receptores de Glutamato/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Proteínas Portadoras/química , Simulación por Computador , Electrofisiología , Glutamatos/farmacología , Células HeLa , Humanos , Técnicas In Vitro , Ácido Kaínico/análogos & derivados , Ácido Kaínico/farmacología , Ligandos , Modelos Moleculares , Datos de Secuencia Molecular , Oocitos , Ácido Quiscuálico/farmacología , Proteínas Recombinantes de Fusión , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transfección , Xenopus laevis , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/farmacologíaRESUMEN
Five cDNAs from the cellulolytic fungi Fusarium oxysporum that code for five distinct cellulase homologues have been cloned and sequenced. The cloning strategy exploited the hydrophobic cluster analysis-based cellulase family classification of Henrissat and Bairoch [Biochem. J. 293 (1993) 781-788] to design degenerate oligodeoxyribonucleotides (oligos) that encoded amino-acid sequences conserved in an intra-family, but not inter-family, manner among cellulases from different species. Polymerase chain reaction (PCR) experiments using F. oxysporum genomic DNA primed with these 'family-specific' oligos were used to rapidly generate PCR fragments which were in turn used to probe cDNA libraries. Two distinct cDNAs coding for cellulase C-family homologues and one cDNA each coding for homologues to the B, F and K families, were isolated in this manner. This approach is an example of the power of multiple sequence analysis to generate cross-species, homology-based probes to rapidly clone homologues in a species of interest.
Asunto(s)
Celulasa/genética , Secuencia Conservada , Fusarium/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular/métodos , ADN de Hongos , Fusarium/enzimología , Datos de Secuencia Molecular , Homología de Secuencia de AminoácidoRESUMEN
The primary sequence of a cDNA encoding a novel transglutaminase from a human prostate cDNA library is reported. The sequence is compared to other known transglutaminases in a multiple alignment. The deduced peptide sequence is 51% identical to a rat prostate transglutaminase.
Asunto(s)
Clonación Molecular , ADN Complementario/química , Próstata/química , Transglutaminasas/genética , Secuencia de Aminoácidos , Composición de Base , Secuencia de Bases , Codón , ADN Complementario/genética , Humanos , Masculino , Datos de Secuencia Molecular , Alineación de Secuencia , Transglutaminasas/químicaRESUMEN
We have cloned and characterized a second form of the human calcitonin receptor from T47D cells. It resembles the clone described by Gorn et al. [J. Clin. Invest. 90:1726-1735 (1992)] except that it lacks a 16-amino acid insert in the putative first intracellular loop. The insert-negative receptor appears to be the most abundant form, and it occurs at a relatively constant level in all expressing tissues. In contrast, the insert-positive receptor is found at low levels in most tissues but its expression levels appear to be much more variable. The insert-negative cDNA was stably expressed in baby hamster kidney cells. Like the endogenous T47D receptor, the recombinant receptor has an equally high affinity for salmon and porcine calcitonin but a 3-4-fold lower affinity for human calcitonin. High concentrations of calcitonin gene-related peptide, rat amylin, secretin, or vasoactive intestinal peptide do not significantly compete with calcitonin for binding to the recombinant receptor. Calcitonin stimulates a cAMP response in both T47D and transfected baby hamster kidney cells. Salmon calcitonin is more potent than human calcitonin for T47D cells, but the two are nearly equipotent for the transfectants. Furthermore, the ED50 for the cAMP response in the transfectants is 10-100-fold lower than in T47D cells. Calcitonin stimulates inositol phosphate turnover and elevates internal calcium levels in the transfectants. This response requires non-physiological levels of calcitonin and is directly correlated with the number of receptors. Lastly, by using a human/rodent somatic cell hybrid panel and in situ hybridization, we localized the human calcitonin receptor gene to chromosome 7.
Asunto(s)
Receptores de Calcitonina/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Calcio/metabolismo , Línea Celular , Mapeo Cromosómico , Cromosomas Humanos Par 7 , Clonación Molecular , Cricetinae , AMP Cíclico/metabolismo , Cartilla de ADN , Humanos , Fosfatos de Inositol/biosíntesis , Ratones , Datos de Secuencia Molecular , Receptores de Calcitonina/metabolismo , Homología de Secuencia de Aminoácido , Distribución Tisular , Células Tumorales CultivadasRESUMEN
Receptors for the major excitatory neurotransmitter glutamate include metabotropic (G protein-coupled) and ionotropic (glutamate-gated ion channel) types. These receptors have large, presumably extracellular, amino-terminal domains. Sensitive sequence analysis techniques indicate that the metabotropic receptor extracellular domain is similar to bacterial periplasmic amino acid binding proteins. A structural model built using the observed similarity predicts a ligand-binding site, and mutants with conservative amino acid substitutions at this site are shown to have reduced ligand affinity. The metabotropic receptor extracellular domain is a member of a family of structural domains linked to a variety of receptor types, including ionotropic glutamate receptors.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Receptores de Glutamato/genética , Receptores de Glutamato/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Bases de Datos Factuales , Predicción , Ligandos , Modelos Moleculares , Datos de Secuencia Molecular , Homología de Secuencia de AminoácidoRESUMEN
Glucagon and the glucagon receptor are a primary source of control over blood glucose concentrations and are especially important to studies of diabetes in which the loss of control over blood glucose concentrations clinically defines the disease. A complementary DNA clone for the glucagon receptor was isolated by an expression cloning strategy, and the receptor protein was expressed in several kidney cell lines. The cloned receptor bound glucagon and caused an increase in the intracellular concentration of adenosine 3', 5'-monophosphate (cAMP). The cloned glucagon receptor also transduced a signal that led to an increased concentration of intracellular calcium. The glucagon receptor is similar to the calcitonin and parathyroid hormone receptors. It can transduce signals leading to the accumulation of two different second messengers, cAMP and calcium.
Asunto(s)
Glucagón/farmacología , Hígado/metabolismo , Receptores de la Hormona Gastrointestinal/fisiología , Transducción de Señal , Secuencia de Aminoácidos , Animales , Calcio/farmacología , Línea Celular , Clonación Molecular , Cricetinae , AMP Cíclico/metabolismo , Glucagón/metabolismo , Riñón , Cinética , Datos de Secuencia Molecular , Ratas , Receptores de la Hormona Gastrointestinal/genética , Receptores de la Hormona Gastrointestinal/metabolismo , Receptores de Glucagón , TransfecciónRESUMEN
Glutamine:fructose-6-phosphate amidotransferase (GFAT) has recently been shown to be an insulin-regulated enzyme that plays a key role in the induction of insulin resistance in cultured cells. As a first step in understanding the molecular regulation of this enzyme the human form of this enzyme has been cloned and the functional protein has been expressed in Escherichia coli. A 3.1-kilobase cDNA was isolated which contains the complete coding region of 681 amino acids. Expression of the cDNA in E. coli produced a protein of approximately 77 kDa and increased GFAT activity 4.5-fold over endogenous bacterial levels. Recombinant GFAT activity was inhibited 51% by UDP-GlcNAc whereas bacterial GFAT activity was insensitive to inhibition by UDP-GlcNAc. On the basis of these results we conclude that: 1) functional human GFAT protein was expressed, and 2) the cloned human cDNA encodes both the catalytic and regulatory domains of GFAT since the recombinant GFAT was sensitive to UDP-GlcNAc. Overall, the development of cloned GFAT molecular probes should provide new insights into the development of insulin resistance by allowing quantitation of GFAT mRNA levels in pathophysiological states such as non-insulin-dependent diabetes mellitus and obesity.
Asunto(s)
ADN/genética , Escherichia coli/genética , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , ADN/aislamiento & purificación , Electroforesis en Gel de Poliacrilamida , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/aislamiento & purificación , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/metabolismo , Humanos , Cinética , Datos de Secuencia Molecular , Peso Molecular , Oligodesoxirribonucleótidos , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Human progalanin cDNA was cloned with polymerase chain reaction techniques. The cDNA sequence predicts that the human form of galanin has a substitution of the glycine residue found at position 30 in other species and thus is likely to retain this residue during posttranslational processing and not be amidated at the COOH terminus. Furthermore, the cDNA sequence predicts three additional amino acid substitutions compared with known galanins. Human galanin was synthesized, and its bioactivity was compared with porcine and rat galanin based on inhibition of insulin release from a glucose-responsive rat insulinoma (RIN) cell line. Human galanin inhibited glucose-stimulated insulin secretion in a dose-dependent manner in RIN cells. Human, porcine, and rat galanin exhibited similar activity with ED50 less than 1 nM.