家鸡G蛋白偶联受体85基因的结构、序列与组织表达分析

G蛋白偶联受体(GPCR)超家族是细胞膜上广泛存在的一类受体,是细胞跨膜信号转导的一类重要受体分子,参与许多生理过程调节。它们中仍有很多至今尚未找到内源性配体,这类受体被称为孤儿型受体。G蛋白偶联受体85(GPR85)是GPCR超家族中孤儿型受体的一员。目前,在非哺乳类脊椎动物中,针对GPR85的研究极少。本研究以家鸡Gallus gallus domesticus为模型,通过反转录PCR和RACE-PCR等方法从脑中克隆到GPR85基因的cDNA全长序列,揭示其基因结构,并用实时荧光定量PCR(qPCR)方法探究了该基因在家鸡各组织中的表达情况。结果显示:家鸡GPR85基因位于1号染色体上,由2个外显子组成,其编码区位于第2个外显子上,长为1 113 bp,可编码1个370个氨基酸的7次跨膜受体蛋白。家鸡GPR85与其他脊椎动物(人Homo sapiens、小鼠Mus musculus、大鼠Rattus norvegicus、热带爪蟾Xenopus tropicalis和斑马鱼Danio rerio)的GPR85具有高度的氨基酸序列一致性(>93%)。qPCR分析发现,GPR85基因mRNA在家鸡全脑、垂体、肾上腺、精巢中有较高表达,而在所检测的其他外周组织中表达极低。本研究首次揭示了家鸡GPR85基因的结构与表达特征,为后续探究GPR85基因在家鸡等非哺乳类中的生理功能奠定基础。     

Cloning, Pharmacology, and Tissue Distribution of G-Protein-Coupled Receptor GPR105 (KIAA0001) Rodent Orthologs

It has recently been shown that UDP-glucose is a potent agonist of the orphan G-protein-coupled receptor (GPCR) KIAA0001. Here we report cloning and analysis of the rat and mouse orthologs of this receptor. In accordance with GPCR nomenclature, we have renamed the cDNA clone, KIAA0001, and its orthologs GPR105 to reflect their functionality as G-protein-coupled receptors. The rat and mouse orthologs show 80% and 83% amino acid identity, respectively, to the human GPR105 protein. We demonstrate by genomic Southern blot analysis that there are no genes in the mouse or rat genomes with higher sequence similarity. Chromosomal mapping shows that the mouse and human genes are located on syntenic regions of chromosome 3. Further analyses of the rat and mouse GPR105 proteins show that they are activated by the same agonists as the human receptor, responding to UDP-glucose and closely related molecules with similar affinities. The mouse and rat receptors are widely expressed, as is the human receptor. Thus we conclude that we have identified the rat and mouse orthologs of the human gene GPR105.     

Identification of relaxin-3/INSL7 as an endogenous ligand for the orphan G-protein-coupled receptor GPCR135

GPCR135, publicly known as somatostatin- and angiotensin-like peptide receptor, is expressed in the central nervous system and its cognate ligand(s) has not been identified. We have found that both rat and porcine brain extracts stimulated 35S-labeled guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) incorporation in cells over-expressing GPCR135. Multiple rounds of extraction, purification, followed by N-terminal sequence analysis of the ligand from porcine brain revealed that the ligand is a product of the recently identified gene, relaxin-3 (aka insulin-7 or INSL7). Recombinant human relaxin-3 potently stimulates GTPgammaS binding and inhibits cAMP accumulation in GPCR135 overexpressing cells with EC50 values of 0.25 and 0.35 nM, respectively. 125I-Relaxin-3 binds GPCR135 at high affinity with a Kd value of 0.31 nM. Relaxin-3 is the only member of the insulin/relaxin superfamily that can activate GPCR135. In situ hybridization showed that relaxin-3 mRNA is predominantly expressed in the dorsomedial ventral tegmental nucleus of the brainstem (aka nucleus incertus), as well as in discrete cells in the lateral periaqueductal gray and in the central gray nucleus. GPCR135 is expressed abundantly in the hypothalamus with discrete expression in the paraventricular nucleus of the hypothalamus and supraoptic nucleus, as well as in the cortex, septal nucleus, and preoptical area. Relaxin-3 has previously been shown to bind and activate the LGR7 relaxin receptor. However, we believe that neuroanatomical colocalization of GPCR135 and relaxin-3, coupled with a clear high affinity interaction, suggest that GPCR135 is the receptor for relaxin-3. The identification of relaxin-3 as the ligand for GPCR135 provides the framework for the discovery of a new brainstem/hypothalamus circuitry.     

Molecular characterization of a novel glycoprotein hormone G-protein-coupled receptor

We report the molecular characterization of a novel G-protein-coupled receptor, GPR48, that resembles proteins in the glycoprotein hormone receptor family. The full-length human GPR48 cDNA is comprised of 951 amino acids. The large extracellular amino terminus of 538 residues is composed of seventeen leucine-rich repeats (LRR). The genomic structure of GPR48 has several features in common with genes in the glycoprotein hormone receptor family. Analogous to these receptors, most of the LRR are encoded on single small exons, and the last exon encodes the seven transmembrane segments. The complete gene spans more than 60 kb with 18 exons and 17 introns. Northern blot analysis demonstrated high expression of GPR48 in the adult human pancreas, with moderate levels of expression in placenta, kidney, brain, and heart. Additionally, this receptor is expressed as early as 7 days post coitus in the mouse, indicating its potential involvement in development.     

An evolutionarily conserved G-protein coupled receptor family, SREB, expressed in the central nervous system

We report here a novel family of G-protein coupled receptor (GPCR) which is extraordinarily conserved among vertebrate species. This family, designated SREB (Super Conserved Receptor Expressed in Brain), consists of at least three members, termed SREB1, SREB2, and SREB3. SREB members share 52-63% amino acid identity with each other and show relatively high similarity to previously known amine amine GPCRs (approximately 25% identity). Amino acid sequence identity between human and rat orthologues is 97% for SREB1 and 99% for SREB3, while the SREB2 sequence is surprisingly completely identical between the species. Furthermore, amino acid sequence of zebrafish SREB2 and SREB3 are 94 and 78% identical to mammal orthologues. Northern blot analysis revealed that SREB members are predominantly expressed in the brain regions and genital organs. Radiation hybrid analysis localized SREB1, SREB2, and SREB3 genes to different human chromosomes, namely 3p21-p14, 7q31 and Xp11, respectively. The high sequence conservation and abundant expression in the central nervous system suggest the existence of undiscovered fundamental neuronal systems consisting of SREB family members and their endogenous ligand(s).     

Cloning of a novel G protein-coupled receptor, SLT, a subtype of the melanin-concentrating hormone receptor

A DNA fragment encoding an amino acid sequence possessing common features to the G protein-coupled receptor (GPCR) superfamily was found in the human genomic sequence, and from this information, the full-length cDNA of a novel GPCR, designated SLT, was cloned from the human hippocampus cDNA library. SLT showed the highest homology to the melanin-concentrating hormone (MCH) receptor, SLC-1 (31.5% identity), and to a lesser extent, to the somatostatin (SST) receptor subtypes. MCH exhibited agonistic behavior when applied to the SLT-expressing CHO cells at subnanomolar doses whereas more than 200 known peptides, including SST and cortistatin, did not. These results indicated that MCH is the cognate ligand of the SLT receptor and that this newly cloned GPCR is the second subtype of the MCH receptor. Quantitative polymerase chain reaction analysis of the SLT gene expression in human tissues showed that the SLT receptor is expressed mainly in brain areas including the cerebral cortex, amygdala, hippocampus, and corpus callosum, as well as in a limited number of peripheral tissues. The distribution of the SLT nearly overlapped that of SLC-1, suggesting that some of the neural functions of MCH may be mediated by both of these receptor subtypes.     

Characterization and Expression of the Human A2a Adenosine Receptor Gene

Abstract: The actions of the neurotransmitter adenosine are mediated by a family of high-affinity, G protein-coupled receptors. We have characterized the gene for the human A2a subtype of adenosine receptor (hA2aR) and determined levels of A2aR mRNA in human brain regions and nonneural tissues. Human genomic Southern blot analysis demonstrates the presence of a single gene encoding the hA2aR located on chromosome 22. Two overlapping cosmids containing the hA2aR gene were isolated from a chromosome 22 library and characterized. Southern blot and sequence analyses demonstrate that the hA2aR gene spans ∼9–10 kb with a single intron interrupting the coding sequence between the regions encoding transmembrane domains III and IV. The sequence of the hA2aR gene diverged from the reported cDNA structure in the 5' untranslated region. This divergence appears to result from an artifact in the construction of the original cDNA library. By northern blot analysis, high expression of the hA2aR gene was identified in the caudate nucleus with low levels of expression in other brain regions. High expression was also seen in immune tissues; lesser A2aR expression was detected in heart and lung. The gene for the A2a subtype of receptor for the neurotransmitter adenosine falls in the class of intron containing G protein-coupled receptor genes. Expression in the basal ganglia is consistent with a role for the hA2aR in motor control. Activation of the A2aR may also regulate immune responses and cardiopulmonary function.     

Molecular cloning of a mineralocorticoid (type I) receptor complementary DNA from rat hippocampus

Rat brain expresses two types of corticosteroid-binding proteins. The type I receptor binds corticosterone with high affinity and is structurally related to the kidney mineralocorticoid receptor (MR), while the type II or classical glucocorticoid receptor binds corticosterone with lower affinity and displays an in vivo preference for dexamethasone. Here we describe the isolation and characterization of a cDNA coding for the MR, from a rat hippocampus cDNA library, by low stringency hybridization to radiolabeled human glucocorticoid receptor cDNA. The nucleotide and deduced amino acid sequence for rat hippocampal MR displays extensive homology to a MR cDNA isolated from human kidney, suggesting that they are orthologous genes. Southern analysis suggests that there is only one gene for the MR, and in vitro expression of the receptor generates a high affinity corticosterone-binding protein. These data provide evidence to support the contention that a single gene gives rise to the MR in renal tissues and type I receptors in the brain.     

Tx基因与Igk基因的同源性研究及其在不同细胞株的表达

本文对以前报道的Ｔｘ基因２．８ｋｂ片段的核苷酸序列与人免疫球蛋白ｋａｐｐａ链Ｃ区基因的核苷酸序列及其编码产物的氨基酸序列进行了同源性比较。结果表明,Ｔｘ基因与ｋａｐｐａ链Ｃ区基因的同源性高达９９．５％以上,编码区的同源性高达１００％。从而提示Ｔｘ基因与ｋａｐｐａ链Ｃ区基因可能是同一种基因。限制性内切酶图谱及Ｓｏｕｔｈｅｒｎ印迹杂交分析,也进一步支持这一观点。本文还报道了ｋａｐｐａ链Ｃ区基因在不同细     

Primary structure and distribution of a novel ryanodine receptor/calcium release channel from rabbit brain.

The complete amino acid sequence of a novel ryanodine receptor/calcium release channel from rabbit brain has been deduced by cloning and sequence analysis of the cDNA. This protein is composed of 4872 amino acids and shares characteristic structural features with the skeletal muscle and cardiac ryanodine receptors. RNA blot hybridization analysis shows that the brain ryanodine receptor is abundantly expressed in corpus striatum, thalamus and hippocampus, whereas the cardiac ryanodine receptor is more uniformly expressed in the brain. The brain ryanodine receptor gene is transcribed also in smooth muscle.     

Isolation and Chromosomal Localization of GPR31, a Human Gene Encoding a Putative G Protein-Coupled Receptor

The screening of a human genomic library with a chemokine receptor-like probe allowed us to obtain a putative member of the G protein-coupled receptor gene (GPCR) family, designated GPR31. Its deduced amino acid sequence encodes a polypeptide of 319 amino acids that shares 25–33% homology with members of the chemokine, purino, and somatostatin receptor gene families. Amino acid sequence comparison reveals that the best match in the protein databases is with the human orphan GPCR called HM74 (33% identity). Southern genomic analysis of the GPR31 gene shows a hybridization pattern consistent with that of a single-copy gene. Using fluorescencein situhybridization, we have determined the chromosomal and regional localization of the GPR31 gene at 6q27. The GPR31 mRNA is expressed at low levels by several human cell lines of different cellular origins. The phylogenetic analysis suggests that the GPR31 receptor may represent a member of a new GPCR subfamily.     

Cloning, expression, and characterization of a gene encoding the human angiotensin II type 1A receptor.

The human angiotensin II (AII) type 1a receptor gene and its upstream control sequence has been cloned from a human leukocyte genomic library. The promoter element CAAT and TATA sequences were found at -602 and -538, respectively, upstream from the translational initiation site. The deduced protein sequence is homologous to rat and bovine AT1a receptors (94.7% and 95.3% identity). The expressed gene exhibited high-affinity AII and Dup753 binding and was functionally coupled to inositol phosphate turnover. Northern analysis of human tissues showed AT1 receptor mRNA expression in placenta, lung, heart, liver, and kidney. Using 5' untranslated and coding sequence as probes in a Southern blot analysis, it was established that another AT1 subtype exists in the human genome.