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Neuropeptide Y (NPY) and the pancreatic polypeptide (PP) are members of the neuropeptide Y family of hormones. They bind to the Y receptors with very different affinities: Whereas PP is highly selective for the Y(4) receptor, NPY displays highest affinites for Y(1), Y(2), and Y(5) receptor subtypes. Introducing the NPY segment 19-23 into PP leads to an increase in affinity at the Y(1) and Y(2) receptor subtypes whereas the exchange of this segment from PP into NPY leads to a large decrease in affinity at all receptor subtypes. PP displays a very stable structure in solution, with the N terminus being back-folded onto the C-terminal alpha-helix (the so-called PP-fold). The helix of NPY is less stable and the N terminus is freely diffusing in solution. The exchange of this segment, however, does not alter the PP-fold propensities of the chimeric peptides in solution. The structures of the phospholipid micelle-bound peptides serving to mimic the membrane-bound species display segregation into a more flexible N-terminal region and a well-defined alpha-helical region. The introduction of the [19-23]-pNPY segment into hPP leads to an N-terminal extension of the alpha-helix, now starting at Pro(14) instead of Met(17). In contrast, a truncated helix is observed in [(19)(-)(23)hPP]-pNPY, starting at Leu(17) instead of Ala(14). All peptides display moderate binding affinities to neutral membranes (K(assoc) in the range of 1.7 to 6.8 x 10(4) mol(-)(1) as determined by surface plasmon resonance) with the differences in binding being most probably related to the exchange of Arg-19 (pNPY) by Glu-23 (hPP). Differences in receptor binding properties between the chimeras and their parental peptides are therefore most likely due to changes in the conformation of the micelle-bound peptides.

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IntroductionG protein-coupled receptors (GPCR) play an important role in signaling cascades, where they participate in the transduction of extracellular signals to intracellular heterotrimeric guanyl nucleotide binding proteins (G proteins). GPCRs are a large superfamily with a structure comprising an extracellular amino terminus, seven transmembrane-spanning α-helices connected by alternating extracellular and intracellular loops, and a cytoplasmic carboxyl terminal region.1 The arrangement of the seven transmembrane-spanning helices and the extracellular domains provides a specific binding site for ligands, which induces a conformational change in the receptor that exposes intracellular regions, which recruit and activate G proteins.The type 1 angiotensin II (AngII) receptor (AT1A) is a 359 amino acid GCPR that mediates the important cardiovascular and homeostatic actions of the peptide hormone angiotensin II. The 54 amino acid intracellular carboxyl terminus (Leu 305 to Glu 359 ) of the AT1A receptor interacts with G proteins 2 and other signaling molecules, 3 indicating a contribution to receptor activation, while the discovery of phosphorylation sites 4 and internalization motifs 5-7 suggest an involvement in receptor regulation. In particular, the proximal region (Leu 305 to Pro 321 ), comprising the first third of the AT1A receptor carboxylterminus, is an important site of complex interactions for receptor function.In previous work, we proposed that the proximal carboxylterminus of the AT1A receptor (residues 305 -320) is an amphipathic α-helix (now referred to as helix VIII), and that this structure may be relevant to receptor activation and internalization on the basis of computer modeling. 6,8,9 In order to investigate the conformation and orientation of the proximal carboxyl terminus, the interaction of a synthetic peptide corresponding to the carboxyl terminus (Leu 305 to Lys 325 ) of the receptor (AT peptide) and its analogue peptide (where lysine residues at 307, 308, 310, and 311 were substituted with norleucine) with a zwitterionic or anionic lipid membrane as a model was examined by surface plasmon resonance (SPR). 8 The AT peptide was shown by SPR to bind with high affinity to a negatively charged lipid membrane via electrostatic interactions.We have already established an evaluation method of the interactions of various peptides to lipid membranes using SPR, 9-15 and demonstrated that the kinetic rate constants and the affinity of membrane interactions can be readily determined by this technique, which in turn provides important insights into the mechanism of peptide-membrane interactions. In the present study, we extended our studies into the membrane binding properties of the AT peptide.Specifically, we examined the membrane-binding properties of a series of peptide analogues of the AT peptide in which particular amino acid residues have been modified to establish their role in the structure and membrane binding. We determined the kinetic rate constants and affinity of these synthetic peptid...