Search

Background: Based on sequence similarity, the superfamily of G protein-coupled receptors (GPRs) can be subdivided into several subfamilies, the members of which often share similar ligands. The sequence data provided by the human genome project allows us to identify new GPRs by in silico homology screening, and to predict their ligands. Results: By searching the human genomic database with known nucleotide receptors we discovered the gene for GPR99, a new orphan GPR. The mRNA of GPR99 was found in kidney and placenta. Phylogenetic analysis groups GPR99 into the P2Y subfamily of GPRs. Based on the phylogenetic tree we propose a new classification of P2Y nucleotide receptors into two subgroups predicting a nucleotide ligand for GPR99. By assaying known nucleotide ligands on heterologously expressed GPR99, we could not identify specifically activating substances, indicating that either they are not agonists of GPR99 or that GPR99 was not expressed at the cell surface. Analysis of the chromosomal localization of all genes of the P2Y subfamily revealed that all members of subgroup "a" are encoded by less than 370 kb on chromosome 3q24, and that the genes of subgroup "b" are clustered on one hand to chromosome 11q13.5 and on the other on chromosome 3q24-25.1 close to the subgroup "a" position. Therefore, the P2Y subfamily is a striking example for local gene amplification. Conclusions: We identified a new orphan receptor, GPR99, with homology to the family of G protein-coupled nucleotide receptors. Phylogenetic analysis separates this family into different subgroups predicting a nucleotide ligand for GPR99. [ABSTRACT FROM AUTHOR]

The two human proteins with a VPS10 domain, SorLA and sortilin, both bind neuropeptides. Searching for other VPS10-domain proteins in the database revealed three new putative human neuropeptide receptors. The new receptors were designated SorCS1, SorCS2 and SorCS3, due to their identical domain composition, which, except for the N-terminal VPS10 domain, differs from that of SorLA and sortilin. Using the databases of the human genome project we elucidated the exon-intron structures of the human VPS10-receptor genes. They contain many short exons, separated by introns, several of which extend over more than 50 kb. The three SorCS genes encompass more than 500 kb of genomic DNA and therefore represent some of the largest known human genes. All these genes map to chromosomal localisations of known genetic diseases, many of them neurological disorders, corresponding to the strong expression of these receptors in the brain. CpG islands are located in the first exon of each of the VPS10-receptor genes and might be involved in developmental or tissue-specific regulation of gene expression. [ABSTRACT FROM AUTHOR]

By exposing different sites of the 'head activator', different sets of antibodies were designed and produced which recognized either the amino or carboxy terminus of the free' head activator', which reacted with 'head activator' in a tissue-fixed conformation, of which bound to the 'head-activator' sequence, if it was part of a larger precursor-like molecule. An enzyme-linked immunosorbent assay (ELISA) was developed to characterize in animal or tissue extracts. A competitive ELISA is described which uses biotin-avidin for enhancement. The assay is sensitive with an antibody specific for the terminus in the range of 0.5-50 fmol, with an antibody specific for the carboxy terminus in the range of 20-400 fmol. The ELISA specific for the amino terminus is 10-times more sensitive than a radio-immunoassay with tritiated 'head activator' [H.Bodenmuller and B. Zachmann (1983) FEBS Lett. 159,237-240]. Previously no radioimmunoassay existed with specificity for the carboxy terminus. [ABSTRACT FROM AUTHOR]

The mouse orthologue of somatostatin and angiotensin-like peptide receptor (SALPR) was amplified from cDNA of the hippocampal cell line HT22. It coded for a protein of 472 amino acids showing 84% sequence identity with human SALPR and 43% with human G-protein-coupled receptor 100 (GPR100). A distinct pattern of expression in brain, spinal cord, and dorsal root ganglia during development and in the mature brain hint at important functions of SALPR for differentiation and maintenance of the nervous system. [Copyright &y& Elsevier]

We report the identification, cloning, and localization of human and mouse orthologues of a new G-protein-coupled receptor with homology to galanin receptors, which we termed galanin-receptor like (GalRL). The genes of GalRL were localized to chromosome 5q32 in human and to 18B3 in mouse. Northern blot analysis revealed expression of GalRL in the central nervous system of human and mouse and in 7- and 15-day-old mouse embryos. Minor levels were found in some peripheral organs and tissues, such as testis, liver, kidney and stomach. In situ hybridization experiments demonstrated predominant expression of GalRL in the central nervous system, with a distinct localization in the habenular complex. In the peripheral nervous system single neurons of sensory ganglia were labeled. During embryonal development the expression was more widespread in the nervous system, where in addition to the dorsal thalamus, hybridization signals were detected in other areas of the brain including the striatum, the locus coeruleus, and several hindbrain nuclei. A weak activation of GalRL by galanin suggests that the endogenous ligand shares structural features with galanin. [Copyright &y& Elsevier]

We identified and cloned the mouse orthologue of human GPR6 as a new member of the lysophospholipid-receptor family. Sphingosine-1-phosphate (S1P) activated GPR6, transiently expressed in frog oocytes or in Chinese hamster ovary (CHO) cells, with high specificity and nanomolar affinity. The GPR6 gene was found to be located on chromosome 10B1 and a single exon coded for the entire open-reading frame. Signal transduction of S1P was inhibited by pertussis toxin, suggesting a coupling of GPR6 to an inhibitory G protein. In CHO cells transfected with GPR6, the sphingosine-kinase pathway mediated Ca(2+) mobilization from internal stores. Apoptotic cell death was induced by serum deprivation or H(2)O(2) treatment and was prevented by S1P in GPR6-, but not in vector-transfected CHO cells. The antiapoptotic effect of S1P required activation of sphingosine kinase and was accompanied by an increase in MAP-kinase phosphorylation.

Recently, a new member of the presenilin family was identified as an aspartyl protease that cleaves signal peptides within hydrophobic domains, and was, therefore, named signal peptide peptidase (SPP). We isolated cDNAs coding for mouse and human orthologues of SPP. The human gene spans 55 kilobases on chromosome 20q11.21. The SPP-protein is encoded in mouse and man by 12 exons. The highly conserved intron/exon-structure in the SPP/presenilin family hints at a common precursor. Northern blot and in situ hybridization analysis revealed a widespread expression of SPP in many tissues. A distinct pattern of expression in the mature murine brain and during development indicates that SPP plays an important role in the establishment and maintenance of the nervous system. We prepared an antiserum against the carboxy-terminal domain of SPP, which is highly conserved between species. It reacted specifically, both in western blots and in immunocytochemical preparations, with SPP from various mammalian origins. The antiserum was used to demonstrate that SPP is oriented in the membrane of the endoplasmic reticulum with its carboxy-terminal tail extending into the cytosol.

The single transmembrane receptor sorLA/LR11 contains binding domains typical for the low-density lipoprotein receptors and a VPS10 domain which, in the related receptor sortilin, binds the neuropeptide neurotensin. SorLA is synthesized as a proreceptor which is processed to the mature form by a furin-like propeptidase. Endogenous sorLA and its hydra homologue HAB bind the neuropeptide head activator (HA). Transiently expressed partial sorLA constructs were investigated for ligand binding. We found that HA binds with nanomolar affinity to the VPS10 domain. The sorLA propeptide also bound to the VPS10 domain, whereas the receptor-associated protein RAP interacted both with the class A repeats and the VPS10 domain. The sorLA propeptide inhibited binding of HA to full-length sorLA and to the VPS10 domain. It also interfered with binding of HA to hydra HAB, which is taken as evidence for a highly conserved tertiary structure of the VPS10 domains of this receptor in hydra and mammals. The propeptide inhibited HA-stimulated mitosis and proliferation in the human neuroendocrine cell line BON and the neuronal precursor cell line NT2. We conclude that sorLA is necessary for HA signaling and function.