PCR was used to isolate nucleotide sequences that may encode novel members of the neuropeptide Y receptor family. By use of a PCR product as a hybridization probe, a full-length human cDNA was isolated that encodes a 375-aa protein with a predicted membrane topology identifying it as a member of the G-protein-coupled receptor superfamily. After stable transfection of the cDNA into human embryonic kidney 293 cells, the receptor exhibited high affinity (Kd = 2.8 nM) for 1251-labeled human pancreatic polypeptide (PP).Competition binding studies in whole cells indicated the following rank order of potency: human PP = bovine PP 2 human [Pro34] peptide YY > rat PP > human peptide VY = human neuropeptide Y. Northern blot analysis revealed that human PP receptor mRNA is most abundantly expressed in skeletal muscle and, to a lesser extent, in lung and brain tissue. A rat cDNA clone encoding a high-affinity PP receptor that is 74% identical to the human PP receptor at the amino acid level was also isolated. These receptor clones will be useful in elucidating the functional role of PP and designing selective PP receptor agonists and antagonists.Pancreatic polypeptide (PP), peptide YY (PYY), and neuropeptide Y (NPY) are C-terminal amidated, 36-aa peptides that constitute the mammalian NPY family (1). PP is produced predominantly by pancreatic islet cells and regulates pancreatic exocrine secretion through a mechanism that is not fully understood but may involve inhibition of vagal input to the pancreas (2, 3). PYY-like immunoreactivity is found in endocrine cells of the lower gastrointestinal tract, and the peptide has been shown to have a variety of effects on gastrointestinal function (4, 5). NPY, which is the most highly conserved of the family members between species (6), is extensively distributed throughout both the central and the peripheral nervous systems (7,8). NPY is colocalized with norepinephrine in postganglionic sympathetic nerves (4) and is a potent vasopressor agent (9). The observations of persistent vasoconstriction after a-adrenergic receptor blockade (10, 11) and elevated levels of NPY in hypertensive patients (10, 12) suggest a possible role for NPY in the pathophysiology of hypertension. In the central nervous system, NPY immunoreactivity and mRNA are prevalent in various hypothalamic nuclei, the cerebral cortex, and the septal-hippocampal system and striatum (5). Important effects of centrally administered NPY include hyperphagia (13), anxiolysis (14), and regulation of pituitary hormone release (15).Receptors for NPY/PYY have been delineated on the basis of pharmacological selectivity exhibited by certain tissues and cell lines. The Y1 receptor exhibits high affinity for NPY, PYY, and C-terminal-substituted variants, including [Leu3t,Pro34]NPY, but low affinity for long C-terminal fragments such as 17). This receptor, which is a member of the G-protein-coupled receptor superfamily, has been cloned (18), and its pharmacological properties in transfected cells are identical to those of Y1 rece...

The agouti-related protein gene (Agrp) is a novel gene implicated in the control of feeding behavior. The hypothalamic expression of Agrp is regulated by leptin, and overexpression of Agrp in transgenic animals results in obesity and diabetes. By analogy with the known actions of agouti, these data suggest a role for the Agrp gene product in the regulation of melanocortin receptors expressed in the central nervous system. The availability of recombinant, highly purified protein is required to fully address this potential interaction. A nearly full-length form of AGRP (MKd5-AGRP) was expressed in the cytosolic or soluble fraction of Escherichia coli and appeared as large intermolecular disulfide-bonded aggregates. Following oxidation, refolding, and purification, this protein was soluble, and eluted as a single symmetric peak on RP-HPLC. Circular dichroism studies indicated that the purified protein contains primarily random coil and beta-sheet secondary structure. Sedimentation velocity studies at neutral pH demonstrated that MKd5-AGRP is monomeric at low micromolar concentrations. Mobility shifts observed using SDS-PAGE under reducing and nonreducing conditions for bacterially expressed and mammalian expressed AGRP were identical, an indication of a similar disulfide structure. The purification to homogeneity of a second, truncated form of AGRP (Md65-AGRP) which was expressed in the insoluble or inclusion body fraction is also described. Both forms act as competitive antagonists of alpha-melanocyte stimulating hormone (alpha-MSH) at melanocortin-3 (MC-3) and melanocortin-4 receptors (MC-4). The demonstration that AGRP is an endogenous antagonist with respect to these receptors is a unique mechanism within the central nervous system, and has important implications in the control of feeding.

The amino acid sequence of a staphylococcal delta haemolysin purified from a canine isolate has been determined by a combination of chemical and mass spectroscopic sequence methods. The toxin molecule consists of 26 amino acid residues and is compared with the sequence of a delta haemolysin purified from a human isolate. The two sequences are found to be homologous although amino acid differences are observed in 9 of the 26 residues. Delta hemolysin Toxin

Neuropeptide Y (NPY), peptide YY (PYY), and pancreatic polypeptide (PP) constitute a trio of structurally related peptides that elicit a variety of physiological responses in targets including the brain, gut, and vascular smooth muscle. These actions are mediated by a family of G protein‐coupled receptors, each of which exhibits a distinct pharmacological fingerprint. This unit describes protocols for identifying and characterizing the pharmacological properties of two NPY receptor subtypes, Y1 and Y2. At least four other members of this receptor family have been described (Y3, Y4, Y5 and y6), and a brief description of these receptors and relevant literature citations are also provided.