Characterization of peptide QRFP (26RFa) and its receptor from amphioxus, Branchiostoma floridae.

A peptide ending with RFamide (Arg-Phe-amide) was discovered independently by three different laboratories in 2003 and named 26RFa or QRFP. In mammals, a longer version of the peptide, 43 amino acids, was identified and found to bind to the orphan G protein-coupled receptor GPR103. We searched the genome database of Branchiostoma floridae (Bfl) for receptor sequences related to those that bind peptides ending with RFa or RYa (including receptors for NPFF, PRLH, GnIH, and NPY). One receptor clustered in phylogenetic analyses with mammalian QRFP receptors. The gene has 3 introns in Bfl and 5 in human, but all intron positions differ, implying that the introns were inserted independently. A QRFP-like peptide consisting of 25 amino acids and ending with RFa was identified in the amphioxus genome. Eight of the ten last amino acids are identical between Bfl and human. The prepro-QRFP gene in Bfl has one intron in the propeptide whereas the human gene lacks introns. The Bfl QRFP peptide was synthesized and the receptor was functionally expressed in human cells. The response was measured as inositol phosphate (IP) turnover. The Bfl QRFP peptide was found to potently stimulate the receptor's ability to induce IP turnover with an EC50 of 0.28nM. Also the human QRFP peptides with 26 and 43 amino acids were found to stimulate the receptor (1.9 and 5.1nM, respectively). Human QRFP with 26 amino acids without the carboxyterminal amide had dramatically lower potency at 1.3µM. Thus, we have identified an amphioxus QRFP-related peptide and a corresponding receptor and shown that they interact to give a functional response.

Identification of positions in the human neuropeptide Y/peptide YY receptor Y2 that contribute to pharmacological differences between receptor subtypes.

The members of the neuropeptide Y (NPY) family are key players in food-intake regulation. In humans this family consists of NPY, peptide YY (PYY) and pancreatic polypeptide (PP) which interact with distinct preference for the four receptors showing very low sequence identity, i.e. Y1, Y2, Y4 and Y5. The binding of similar peptides to these divergent receptors makes them highly interesting for mutagenesis studies. We present here a site-directed mutagenesis study of four amino acid positions in the human Y2 receptor. T(3.40) was selected based on sequence alignments both between subtypes and between species and G(2.68), L(4.60) and Q(6.55) also on previous binding studies of the corresponding positions in the Y1 receptor. The mutated receptors were characterized pharmacologically with the peptide agonists NPY, PYY, PYY(3-36), NPY(13-36) and the non-peptide antagonist BIIE0246. Interestingly, the affinity of NPY and PYY(3-36) increased for the mutants T(3.40)I and Q(6.55)A. Increased affinity was also observed for PYY to Q(6.55)A. PYY(3-36) displayed decreased affinity for G(2.68)N and L(4.60)A whereas binding of NPY(13-36) was unaffected by all mutations. The antagonist BIIE0246 showed decreased affinity for T(3.40)I, L(4.60)A and Q(6.55)A. Although all positions investigated were found important for interaction with at least one of the tested ligands the corresponding positions in hY1 seem to be of greater importance for ligand binding. Furthermore these data indicate that binding of the agonists and the antagonist differs in their points of interaction. The increase in the binding affinity observed may reflect an indirect effect caused by a conformational change of the receptor. These findings will help to improve the structural models of the human NPY receptors.

Internalization of pancreatic polypeptide Y4 receptors: correlation of receptor intake and affinity.

Unlike neuropeptide Y receptors, the pancreatic polypeptide Y4 receptors display considerable differences in sequence and ligand-binding affinity across mammalian species. This could produce different receptor turnover rates in the same cellular membrane environment. Comparing rat, human and guinea-pig Y4 receptors expressed in Chinese hamster ovary (CHO) cells (K(d) with human pancreatic polypeptide 14, 45 and 116 pM, respectively), we indeed found human pancreatic polypeptide internalization in the rank order of receptor affinities. A large fraction of the internalized human pancreatic polypeptide, similar across the Y4 species, was associated with secondary endosomes (density approximately 1.05 in Percoll gradients) and lysosomes (density approximately 1.11). For all Y4 receptors examined, this intake was potently and selectively inhibited by cholesterol-complexing polyene antibiotic filipin III and also by clathrin lattice formation inhibitor, phenylarsine oxide. Internalization differences found across Y4 receptor species to a degree compare with those observed for the cloned guinea-pig neuropeptide Y Y1 and human neuropeptide Y Y5 receptors and, generally, support ligand-binding affinities as important determinants of internalization for neuropeptide receptors.

Pancreatic polypeptide receptors: affinity, sodium sensitivity and stability of agonist binding.

Cloned rat, human and guinea-pig Y4 pancreatic polypeptide (PP) receptors expressed in Chinese hamster ovary (CHO) cells, as well as the rabbit Y4-like PP receptor, show a selective sensitivity to Na+ over K+ ion in PP attachment, but little sensitivity to Na+ in dissociation of bound PP peptides. Agonist binding to Y4 receptors of intact CHO cells also shows much greater sensitivity to Na+ over K+, and a tenacious attachment of the bound agonist. Binding sensitivity to K+ is greatly enhanced upon receptor solubilization. Pancreatic polypeptide sites also show large sensitivity to modulators of Na+ transport such as N5-substituted amilorides and to RFamides, as different from Y1 or Y2 receptors. Thus, PP binding is modulated by cation-induced changes in site environment (with selectivity for Na+) and ultimately results in a blocking attachment. This would support receptor operation in the presence of ion gradients, as well as prolonged agonist-delimited signaling activity (which can include partial antagonism). Also, this could point to an evolutionary adaptation enabling small numbers of PP receptors to perform extensive metabolic tasks in response to low agonist signals.