Neuropeptide Y (NPY) is one of the most abundant neuropeptides in the mammalian nervous system and exhibits a diverse range of important physiological activities, including effects on psychomotor activity, food intake, regulation of central endocrine secretion, and potent vasoactive effects on the cardiovascular system. Two major subtypes of NPY receptor (Y1 and Y2) have been defined by pharmaclgical criteria. We report here the molecular cloning of a cDNA sequence encoding a human NPY receptor and the corrected sequence for a rat homologue. Analysis ofthis sequence confirms that the receptor is a member of the G protein-coupled receptor superfamily. When expressed in Chinese hamster ovary (CHO) or human embryonic kidney (293) cells, the receptor exhibits the characteristic ligand specificity of a Y1 type of NPY receptor. In the 293 cell line, the receptor is coupled to a pertussis toxinsensitive G protein that mediates the inhibition of cyclic AMP accumulation. In the CHO cell line, the receptor is coupled not to the inhibition of adenylate cyclase but rather to the elevation of intraceflular calcium. These results demonstrate that second messenger coupling of the NPY-Y1 receptor is cell type specific, depending on the specific repertoire of G proteins and effector systems present in any cell type.Neuropeptide Y (NPY), a 36-amino acid peptide, is an important regulator in both the central and peripheral nervous systems (1). NPY is highly conserved in primary structure between species, as the sequences of human, rat, rabbit, and guinea pig are identical and differ from the porcine sequence by only a single amino acid (2). NPY also shares close sequence homology and a common tertiary structure with a family of peptides which include peptide YY (PYY) and pancreatic polypeptide (PP) This G protein complex in turn activates a variety of second messenger systems, including a decrease in cyclic AMP and an increase in intracellular calcium (10). However, there are reports of NPY receptors coupled to phosphoinositol metabolism, suggesting the existence ofadditional receptor subtypes and/or multiple functions for the Y1 and Y2 subtypes (6, 11).We report here the molecular cloning of a cDNA sequence encoding a human NPY receptor,* which exhibits the characteristic ligand specificity of a Y1 receptor. When expressed in different cell lines, the receptor couples via pertussis toxin-sensitive G proteins to different second messenger systems.MATERIAL AND METHODS Nucleotide Sequence Determination. Total RNA (3 pug) from rat brain was used as a template to synthesize random primed single-stranded cDNA. The cDNA was used in a polymerase chain reaction (PCR) together with the oligonucleotide primers, which correspond to positions 672-584 and 48-78 in the rat cDNA clone FC5 (12). PCR conditions: 30 cycles at 950C for 1 min, 630C for 2 min, and 720C for 1 min. The reaction product was digested with EcoRI and Pst I, gel purified, and subcloned for sequencing in the Bluescript vector (Stratagene) for confirmation of the seq...

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Dopamine, a major neurotransmitter in the mammalian nervous system, exerts its physiological effects through receptors of the G-protein-coupled receptor superfamily. Two major classes of dopamine receptor, D1 and D2, are distinguishable by both biochemical and pharmacological criteria. D1 receptors activate adenylyl cyclase, whereas the D2 class of receptors inhibits this second messenger system. Two subtypes of the human dopamine D2 receptor are generated by alternate splicing of the RNA transcript of a single gene. These two forms, termed D2A (long) and D2B (short), differ by the insertion of 29 amino acids within the putative third cytoplasmic loop, an intracellular domain thought to have a role in coupling this class of receptors to particular second messenger systems. We report here that the D2A and D2B structural subtypes are also functionally distinct. Expression of the two subtypes in a fibroblast cell line revealed that while occupation of both receptors leads to an increase in cytosolic free calcium concentration, they differ in their capacity to inhibit cAMP production. At physiological dopamine concentrations, the D2B-mediated inhibition of calcitonin gene-related peptide-stimulated cAMP accumulation is almost double the response mediated by the D2A subtype. Furthermore, the D2B subtype can maximally attenuate cAMP accumulation by up to 85%, whereas the D2A subtype is less effective, maximally inhibiting cAMP accumulation by only 64%. The D2A and D2B subtypes, thus, constitute functionally distinct forms of the dopamine receptor that can couple to multiple intracellular signalling pathways.

Phosphate (Po) reabsorption in renal proximal tubules involves Na+/P1 cotransport across the brush border membrane; its transport rate is influenced by the Na+-coupled transport of other solutes as well as by pH. In the present study, we have expressed a cloned rat renal brush border membrane Na+/P1 cotransporter (NaPI-2) in Xenopus laevis oocytes and have analyzed its electrophysiologic properties in voltage-and current-clamp studies. Addition of Pi to Na+-containing superhisates resulted in a depolarization of the membrane potential and, in voltage-damped oocytes, in an inward current (Ip). An analysis of the Na+ and/or Pi concentration dependence of Ip suggested a Na+/P1 stoichiometry of 3:1. Ip was increased by increasing the pH ofthe superfusate; this phenomenon seems to be mainly related to a lowering ofthe affinity for Na+ interaction by increasing H+ concentration.The present data suggest that known properties of Pi handling at the tubular/membrane level are "directly" related to specific characteristics of the transport molecule (NaPi-2) Renal Na+/Pi cotransport has been extensively characterized in studies on cortical brush border membrane vesicles (for review, see refs. 1-3; for examples, see refs. 5-7 and 9-17); these studies provided evidence for a Na+/Pi stoichiometry exceeding unity and that the pH dependence of tubular Pi reabsorption might be related to multiple factors, among them strong pH effects on Na+ interaction (5) and some preferential transport of divalent Pi (6,7,10,11).Kinetic studies on brush border membrane Na+/Pi cotransport provided evidence for a heterogeneity/multiplicity of brush border membrane Na+/Pi cotransport systems (16,17 ref. 19) renal Na+/Pi cotransporters have been identified by using the Xenopus laevis expression cloning system; the proximal tubular and brush border location of NaPi-1-and NaPi-2-related transport systems could be documented at the mRNA as well as at the protein level (20)(21)(22). NaPi-1 is not homologous to NaPi-2 or NaPi-3, but the latter two are highly homologous to each other (18,19). Although, NaPi-1 and NaPi-2/3 genes have not been identified within the same species, they might be related to the abovementioned heterogeneity/multiplicity of tubular Na+/Pi cotransporters. Brush border membrane Na+/Pi cotransport in the kidney seems to be different from brush border membrane Na+/Pi cotransport in the small intestine; most strikingly, the rate of small-intestine Na+/P, cotransport is increased by lowering of extravesicular pH (23). Also, use of molecular tools such as cDNA probes and specific antibodies gave no evidence for a structural similarity between renal and intestinal Na+/P1 cotransporters (J.B. and H.M., unpublished work).The above-mentioned tools have been used to study phys- . Further, the content of NaPi-2-related mRNA and protein is reduced in accordance with reduced transport activity in a hypophosphatemic mouse model (Hyp mouse; ref. 26). These observations suggest that NaPi-2-related transport activity is physiologically...