Lee F. Kolakowski

Two period Homologs: Circadian Expression and Photic Regulation in the Suprachiasmatic Nuclei

We have characterized a mammalian homolog of the Drosophila period gene and designated it Per2. The PER2 protein shows >40% amino acid identity to the protein of another mammalian per homolog (designated Per1) that was recently cloned and characterized. Both PER1 and PER2 proteins share several regions of homology with the Drosophila PER protein, including the protein dimerization PAS domain. Phylogenetic analysis supports the existence of a family of mammalian per genes. In the mouse, Per1 and Per2 RNA levels exhibit circadian rhythms in the SCN and eyes, sites of circadian clocks. Both Per1 and Per2 RNAs in the SCN are increased by light exposure during subjective night but not during subjective day. The results advance our knowledge of candidate clock elements in mammals.

Melatonin receptors are for the birds: Molecular analysis of two receptor subtypes differentially expressed in chick brain

Two receptors (CKA and CKB) of the G protein-coupled melatonin receptor family were cloned from chick brain. CKA encodes a protein that is 80% identical at the amino acid level to the human Mel1a melatonin receptor and is thus designated the chick Mel1a melatonin receptor. CKB encodes a protein that is 80% identical to the Xenopus melatonin receptor and defines a new receptor subtype, the Mel1c melatonin receptor, which is distinct from the Mel1a and Mel1b melatonin receptor subtypes. A melatonin receptor family consisting of three subtypes is supported by PCR cloning of distinct melatonin receptor fragments from Xenopus and zebrafish. Expression of CKA and CKB results in similar ligand binding and functional characteristics. The widespread distribution of CKA and CKB mRNA in brain provides a molecular substrate for the profound actions of melatonin in birds.

Molecular characterization and expression of cloned human galanin receptors GALR2 and GALR3.

Abstract: Galanin is a 29‐ or 30‐amino acid peptide with wide‐ranging effects on hormone release, feeding behavior, smooth muscle contractility, and somatosensory neuronal function. Three distinct galanin receptor (GALR) subtypes, designated GALR1, 2, and 3, have been cloned from the rat. We report here the cloning of the human GALR2 and GALR3 genes, an initial characterization of their pharmacology with respect to radioligand binding and signal transduction pathways, and a profile of their expression in brain and peripheral tissues. Human GALR2 and GALR3 show, respectively, 92 and 89% amino acid sequence identity with their rat homologues. Radioligand binding studies with 125I‐galanin show that recombinant human GALR2 binds with high affinity to human galanin (KD = 0.3 nM). Human GALR3 binds galanin with less affinity (IC50 of 12 nM for porcine galanin and 75 nM for human galanin). Human GALR2 was shown to couple to phospholipase C and elevation of intracellular calcium levels as assessed by aequorin luminescence in HEK‐293 cells and by Xenopus melanophore pigment aggregation and dispersion assays, in contrast to human GALR1 and human GALR3, which signal predominantly through inhibition of adenylate cyclase. GALR2 mRNA shows a wide distribution in the brain (mammillary nuclei, dentate gyrus, cingulate gyrus, and posterior hypothalamic, supraoptic, and arcuate nuclei), and restricted peripheral tissue distribution with highest mRNA levels detected in human small intestine. In comparison, whereas GALR3 mRNA was expressed in many areas of the rat brain, there was abundant expression in the primary olfactory cortex, olfactory tubercle, the islands of Calleja, the hippocampal CA regions of Ammons horn, and the dentate gyrus. GALR3 mRNA was highly expressed in human testis and was detectable in adrenal gland and pancreas. The genes for human GALR2 and 3 were localized to chromosomes 17q25 and 22q12.2–13.1, respectively.

NOVEL GPCRS AND THEIR ENDOGENOUS LIGANDS : EXPANDING THE BOUNDARIES OF PHYSIOLOGY AND PHARMACOLOGY

Nearly all molecules known to signal cells via G proteins have been assigned a cloned G-protein-coupled-receptor (GPCR) gene. This has been the result of a decade-long genetic search that has also identified some receptors for which ligands are unknown; these receptors are described as orphans (oGPCRs). More than 80 of these novel receptor systems have been identified and the emphasis has shifted to searching for novel signalling molecules. Thus, multiple neurotransmitter systems have eluded pharmacological detection by conventional means and the tremendous physiological implications and potential for these novel systems as targets for drug discovery remains unexploited. The discovery of all the GPCR genes in the genome and the identification of the unsolved receptor-transmitter systems, by determining the endogenous ligands, represents one of the most important tasks in modern pharmacology.

Cloning of a melatonin-related receptor from human pituitary.

We have cloned an orphan G protein‐coupled receptor from a human pituitary cDNA library using a probe generated by PCR. The cDNA, designated H9, encodes a protein of 613 amino acids that is 45% identical at the amino acid level to the recently cloned human Mel1a and Mel1b melatonin receptors. Structural analyses of the encoded protein and its gene, along with phylogenetic analysis, further show that H9 is closely related to the G protein‐coupled melatonin receptor family. Unusual features of the protein encoded by H9 include a lack of N‐linked glycosylation sites and a carboxyl tail >300 amino acids long. H9 transiently expressed in COS‐1 cells did not bind [125I]melatonin or [3H]melatonin. H9 mRNA is expressed in hypothalamus and pituitary, suggesting that the encoded receptor and its natural ligand are involved in neuroendocrine function.

Characterization of a human gene related to genes encoding somatostatin receptors

We report the identification of a gene, named SLC‐1 1, encoding a novel G protein‐coupled receptor (GPCR). A customized search procedure of a database of expressed sequence tags (dbEST) retrieved a human cDNA sequence that partially encoded a GPCR. A genomic DNA fragment identical to the cDNA was obtained and used to screen a library to isolate the full‐length coding region of the gene. This gene was intronless in its open reading frame, and encoded a receptor of 402 amino acids, and shared −40% amino acid identity in the transmembrane (TM) regions to the five known human somatostatin receptors. Northern blot analysis revealed that SLC‐1 is expressed in human brain regions, including the forebrain and hypothalamus. Expression in the rat was highest in brain, followed by heart, kidney, and ovary. Expression of SLC‐1 in COS‐7 cells failed to show specific binding to radiolabelled Tyr1‐somatostatin‐14, naloxone, bremazocine, 1,3‐di(2‐tolyl)‐guanidine (DTG), or haloperidol. A repeat polymorphism of the form (CA) n was discovered in the 5′‐untranslated region (UTR) of the gene and SLC‐1 was mapped to chromosome 22, q13.3.

Cloning and chromosomal mapping of four putative novel human G-protein-coupled receptor genes.

We report the discovery of four novel human putative G-protein-coupled receptor (GPCR) genes. Gene GPR20 was isolated by amplifying genomic DNA with oligos based on the opioid and somatostatin related receptor genes and subsequent screening of a genomic library. Also, using our customized search procedure of a database of expressed sequence tags (dbEST), cDNA sequences that partially encoded novel GPCRs were identified. These cDNA fragments were obtained and used to screen a genomic library to isolate the full-length coding region of the genes. This resulted in the isolation of genes GPR21, GPR22 and GPR23. The four encoded receptors share significant identity to each other and to other members of the receptor family. Northern blot analysis revealed expression of GPR20 and GPR22 in several human brain regions while GPR20 expression was detected also in liver. Fluorescence in situ hybridization (FISH) was used to map GPR20 to chromosome 8q, region 24.3-24.2, GPR21 to chromosome 9, region q33, GPR22 to chromosome 7, region q22-q31.1, and GPR23 to chromosome X, region q13-q21.1.

Cloning, chromosome localization, expression, and characterization of an Src homology 2 and pleckstrin homology domain-containing insulin receptor binding protein hGrb10γ.

hGrb10α (previously named Grb-IR) is a Src-homology 2 domain-containing protein that binds with high affinity to the tyrosine-phosphorylated insulin receptor and insulin-like growth factor-1 receptor. At least two isoforms of human Grb10, (hGrb10α and hGrb10β), which differ in the pleckstrin homology (PH) domain and the N-terminal sequence, have previously been identified in insulin target tissues such as human skeletal muscle and fat cells. Here we report the cloning of the third isoform of the hGrb10 family (hGrb10γ) from human skeletal muscle and its localization to human chromosome 7. We have also determined the human chromosome localization of Grb7 to 17q21-q22 and Grb14 to chromosome 2. hGrb10γ contains an intact PH domain and an N-terminal sequence that is present in hGrb10α but absent in hGrb10β. RNase protection assays and Western blot analysis showed that hGrb10α and hGrb10γ are differentially expressed in insulin target cells including skeletal muscle, liver, and adipocyte cells. hGrb10γ is also expressed in HeLa cells and various breast cancer cell lines. The protein bound with high affinity to the insulin receptor in cells, and the interaction was dependent on the tyrosine phosphorylation of the receptor. hGrb10γ also underwent insulin-stimulated membrane translocation and serine phosphorylation. hGrb10γ phosphorylation was inhibited by PD98059, a specific inhibitor of mitogen-activated protein kinase kinase, and wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase. Taken together, our data suggest that hGrb10 isoforms are potential downstream signaling components of the insulin receptor tyrosine kinase and that the PH domain may play an important role in the involvement of these isoforms in signal transduction pathways initiated by insulin and other growth factors.

A novel gene codes for a putative G protein‐coupled receptor with an abundant expression in brain

Following the cloning of the dopamine receptors we continued a search of the human genome for related genes. We searched an EST data base and discovered cDNA fragments encoding novel G protein‐coupled receptor genes. The available GenBank sequence of one of these EST fragments showed that it encoded a receptor with closest similarity to the D2 dopamine and adrenergic receptors. This cDNA was used to isolate the gene (GPR19), and the encoded receptor also demonstrated similarity with the neuropeptide Y receptor. The gene was mapped to chromosome 12, in region p13.2–12.3. Northern blot analysis revealed expression of GPR19 in peripheral regions, and brain regions significantly overlapping with the D2 receptor gene expression. A sequence of the rat orthologue of GPR19 was obtained and in situ hybridization analysis demonstrated a very abundant expression in rat brain.

Chromosomal localization1 of GPR48, a novel glycoprotein hormone receptor like GPCR, in human and mouse with radiation hybrid and interspecific backcross mapping

We report the chromosomal localization in both mouse and human of a novel G-protein-coupled receptor, GPR48, which resembles glycoprotein hormone receptors, that may be implicated in Wilms tumor deletion syndromes such as WAGR. This receptor forms a novel sub-family of glycoprotein hormone-like GPCRs. We have mapped this receptor to human chromosome 11p14→p13 by several approaches, including radiation hybrid and interspecific backcross mapping, and show that GPR48 is close to BDNF. This data differs from the recently published mapping of LGR4 (5q34→q35.1) (Hsu et al., 1998). Additionally, we show that Gpr48 and Bdnf are tightly linked on mouse chromosome 2, in a region with conserved synteny to human 11p14→p13.