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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 125l-galanin show that recombinant human GALR2 binds with high affinity to human galanin (K 0 = 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 distritution 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 01-factory cortex, olfactory tubercie, the islands of Calleja, the hippocampal CA regions of Ammon's 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. Key Words: Galanin receptor-G protein-coupled receptor-Cloning.

We have shown in a previous study that desensitization and internalization of the human dopamine D 1 receptor following short-term agonist exposure are mediated by temporally and biochemically distinct mechanisms. In the present study, we have used site-directed mutagenesis to remove potential phosphorylation sites in the third intracellular loop and carboxyl tail of the dopamine D 1 receptor to study these processes. Mutant D 1 receptors were stably transfected into Chinese hamster ovary cells, and kinetic parameters were measured. Mutations of Ser/Thr residues to alanine in the carboxyl tail demonstrated that the single substitution of Thr-360 abolished agonistinduced phosphorylation and desensitization of the receptor. Isolated mutation of the adjacent glutamic acid Glu-359 also abolished agonist-induced phosphorylation and desensitization of the receptor. These data suggest that Thr-360 in conjunction with Glu-359 may comprise a motif necessary for GRK2-mediated phosphorylation and desensitization. Agonist-induced internalization was not affected with mutation of either the Thr-360 or the Glu-359 residues. However, receptors with Ser/Thr residues mutated in the distal carboxyl tail (Thr-446, Thr-439, and Ser-431) failed to internalize in response to agonist activation, but were able to desensitize normally. These results indicate that agonist-induced desensitization and internalization are regulated by separate and distinct serine and threonine residues within the carboxyl tail of the human dopamine D 1 receptor. Dopamine D 1 receptors belong to the family of G proteincoupled receptors (GPCRs 1 ) and mediate their physiological response by coupling with stimulatory G proteins, which in turn activate adenylyl cyclase and increase levels of intracellular cAMP (1). Desensitization of GPCRs is defined as a loss of responsiveness to agonist treatment following prolonged agonist exposure (2, 3). In the dopamine D 1 receptor, we have shown that following short-term agonist exposure, desensitization and internalization were differentially regulated, both biochemically and temporally (4). Rapid desensitization results from receptor uncoupling from G protein following phosphorylation of intracellular domains of the receptor and subsequent internalization of receptor from the cell surface (5). The family of G protein-coupled receptor kinases (GRKs) and second messenger-dependent kinases, such as cAMP-dependent protein kinase (PKA), are serine/threonine kinases that function to uncouple the receptor from G protein (5-8). GRKs additionally promote the interaction of the receptor with arrestin proteins, which recruit the receptors to clathrin-coated pits for subsequent internalization (9).The ␤ 2 -adrenergic receptor is closely related structurally and functionally to the dopamine D 1 receptor, and the definition of the precise sites mediating desensitization occurred in a stepwise manner. Initial studies identified a region of the ␤ 2 -adrenergic receptor carboxyl tail that was involved in agonist-induced desensitization...

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