Pancreatic polypeptide receptors: affinity, sodium sensitivity and stability of agonist binding¹

Abstract

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.

Introduction

Pancreatic polypeptides (PPs), mainly produced in the gut, are a group of neuropeptide Y (NPY) and peptide YY (PYY) -related peptides. Full-length representatives of all three groups are 36-residue peptides containing numerous tyrosines (hence the practice of labeling all NPY, PYY and PP molecules as ‘Y peptides’). The pancreatic polypeptides were discovered and characterized [48] before NPY [101] and PYY [100]. However, research over the last decade reveals that pancreatic polypeptides are evolutionarily much younger than NPY or PYY, the first PP gene deriving from a PYY gene at least 130 Myr after emergence of the ancestral PYY peptide, which should have occurred about 500 Myr ago [51]. Pancreatic polypeptides retain up to 50% overall residue identity to NPY or PYY, and a strong similarity in the C-terminal section (see Fig. 1). However, residues 1–20 of PP molecules differ appreciably from those in NPY or PYY. This apparently results in much lower PP interactivity with membrane constituents, in particular with membrane phospholipids, which from studies with model lipids would avidly associate with NPY [3], [62]. This greatly reduces the ability of pancreatic polypeptides to traverse the membranes independent of association with carriers such as the corresponding specific receptors, resulting in a negligible non-saturable uptake of the peptides [80]. The PPs hence do not readily pass the blood-brain barrier [107], as different from NPY, which displays non-saturable transport in both directions [43], [44]. Incidentally, this also results in much lower non-specific binding of PPs to subcellular membranes or intact cells compared to NPY, and even to PYY, peptides.

The mammalian high-affinity pancreatic polypeptide receptors cloned and sequenced to date include rat [58], mouse [34], human [27], guinea-pig [24] and porcine [111] Y4 species. The mammalian Y4 receptors show up to 25% difference in alignment of primary structure, which is considerably in excess of sequence variation encountered across mammalian Y1 receptors (<7%) and Y2 receptors (<9%) (see e.g. [111]). Across the field of Y receptors, the Y4 receptors have apparently diversified the most, which may indicate greater functional specialization of pancreatic polypeptides vs. the cross-species uniformity of NPY messaging in vertebrates [50], [51]. Distinct less selective PP receptors that also bind NPY and PYY have been characterized in the fish Brachidanio rerio[6], [97], but their pharmacology and physiology needs additional study. Some of these fish molecules might represent distant precursors of Y1 group receptors in higher ectotherms and endotherms.

Regulation of physiological processes by pancreatic polypeptides appears to center on targets outside the blood-brain barrier, which is not readily penetrated by PPs. Likewise, PP receptors identified thus far are largely in the gut [25], [31], and in brain areas accessible to peptides present in general circulation [64], [106], [107]. However, we found significant numbers of PP receptors especially in the kidney [83] and also in the hypothalamus [84] of the rabbit, as well as in the estrogen-induced rat anterior pituitary prolactinoma [79]. These sites were originally thought to be Y5-like, but subsequent comparisons indicated a greater similarity to Y4 receptors ( [80] and this review). Sites responding to avian PPs and identified in the chick [40] have also been reported in the mammal [1], [40], [41].

In view of past findings showing regulation of ion balance by NPY/PYY/PP peptides and receptors [9], [19], [20], as well as the highly selective sensitivity of PP binding to modulators of sodium transport, and to sodium ion itself [79], [80], [81], [82], [84], [85], this review is looking especially into ion regulation of the binding of pancreatic polypeptides to PP receptors. This is supplemented by description of basic features of PP peptides and their probable binding epitopes, as well as of Y4 receptors viewed in the same context.