Cloned human neuropeptide Y receptor couples to two different second messenger systems.

Abstract

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...

NPY Y1 Receptors Differentially Modulate GABAA and NMDA Receptors via Divergent Signal-Transduction Pathways to Reduce Excitability of Amygdala Neurons

“…All NPY receptors have been shown to mediate their responses through G i/o proteins, which inhibit the accumulation of cAMP (Bard et al, 1995;Gerald et al, 1995Gerald et al, , 1996Herzog et al, 1992;Larhammar et al, 1992;Lundell et al, 1995;Mullins et al, 2000). Several additional intracellular signaling pathways of NPY have been reported in peripheral tissues or cell lines.…”

“…Neuropeptide Y (NPY) is widely distributed in the central and peripheral nervous system (Herzog et al, 1992;Tatemoto et al, 1982), where it binds to a family of G-coupled receptors. To date, five NPY receptor subtypes have been cloned (Y 1 , Y 2 , Y 4 , Y 5 , and Y 6 ) from different species (Michel et al, 1998).…”

Neuropeptide Y (NPY) and peptide YY (PYY) effects in the epididymis of the guinea‐pig: evidence of a pre‐junctional PYY‐selective receptor

“…These peptides interact with at least six di erent receptor subtypes (Y 1 ± Y 6 ); cDNAs for Y 1 -, Y 2 -, Y 4 -, Y 5 -receptors and a sixth related receptor have been cloned (Herzog et al, 1992;Larhammar et al, 1992;Bard et al, 1995;Gerald et al, 1995;1996;Lundell et al, 1995;Rose et al, 1995;Gehlert et al, 1996;Gregor et al, 1996;Matsumoto, et al, 1996;Weinberg et al, 1996) and share homology with the superfamily of G protein-coupled receptors. NPY and PYY are approximately equipotent in assays of cultured cells expressing Y 1 , Y 2 and Y 5 receptors (Gerald et al, 1996).…”

Solution Structure of Neuropeptide Tyrosine 13–36, a Y2 Receptor Agonist, as Determined by NMR

“…All investigations agree on the general structure of NPY which is folded in its center forming a hairpin structure with the N-and C-terminal segments in close proximity. The N-terminal polyproline segment (residues 1 -13) is generally disordered and the C-terminal segment (residues 15 -36) is generally helical, [7][8][9][10][11].…”