Peptide ligand binding properties of the corticotropin-releasing factor (CRF) type 2 receptor: pharmacology of endogenously expressed receptors, G-protein-coupling sensitivity and determinants of CRF₂ receptor selectivity

Abstract

The CRF₂ receptor is involved in stress responses, cardiovascular function and gastric motility. Endogenous agonists (urocortin (UCN) 2, UCN 3) and synthetic antagonists (astressin₂-B, antisauvagine-30) are selective for CRF₂ over the CRF₁ receptor. Peptide ligand binding properties of the CRF₂ receptor require further investigation, including ligand affinity for endogenously expressed receptors, the effect of receptor–G-protein coupling on ligand affinity, and the molecular basis of ligand selectivity. Ligand affinity for rat CRF_2(a) in olfactory bulb and CRF_2(b) in A7r5 cells was similar to that for the cloned human CRF_2(a) receptor (within three-fold), except for oCRF (9.4- and 5.4-fold higher affinity in olfactory bulb and A7r5 cells, respectively). Receptor–G-protein uncoupling reduced agonist affinity only 1.2- to 6.5-fold (compared with 92–1300-fold for the CRF₁ receptor). Ligand selectivity mechanisms were investigated using chimeric CRF₂/CRF₁ receptors. The juxtamembrane receptor domain determined selectivity of antisauvagine-30, the N-terminal-extracellular domain contributed to selectivity of UCN 3, and both domains contributed to selectivity of UCN 2 and astressin₂-B. Therefore ligands differ in the contribution of receptor domains to their selectivity, and CRF₂-selective antagonists bind the juxtamembrane domain. These findings will be important for identifying the CRF₂ receptor in tissues and for developing ligands targeting the receptor, both of which will be useful in identifying the emerging physiological functions of the CRF₂ receptor.

Introduction

The corticotropin-releasing factor (CRF) type 2 receptor is a secretin-family G-protein-coupled receptor (GPCR) [31], [32], [39], [43], [53]. Three splice variants have been identified, differing in size and sequence within the extracellular N-terminal domain. Physiological functions of CRF₂ receptors are currently being identified. CRF_2(a) is the most predominant splice variant in the mammalian brain [8], [39], [55]. Proposed central actions of the CRF₂ receptor include behavioral responses to stress and anxiety [3]. Sites of CRF_2(b) expression include the gastrointestinal tract and heart, and CRF_2(b) likely mediates cardiovascular responses in rodents [2], [6], [31], [43] CRF_2(c) has only been identified in humans and is expressed in limbic regions of the CNS [32]. A number of peptide ligands (38–41 amino acids) potently bind and activate the CRF₂ receptor [12]. Urocortin (UCN) I, which also potently activates the related CRF₁ receptor [9], [11], [58], is potentially involved in anxiety responses and hearing [3], [56], [57]. UCN 2 [25], [48] likely activates the CRF₂ receptor in its cardiac function [2]. UCN 3 is expressed in discrete areas of the brain and pancreatic beta cells [25], [35], [36]. The CRF₂ receptor is also potently activated by the amphibian peptide sauvagine [41]. CRF [54], the principle regulator of endocrine stress responses via the CRF₁ receptor, activates the CRF₂ receptor less potently. All of these peptides share homology of amino acid sequence [12]. Two peptide antagonists have been developed, by truncation and sequence modification of sauvagine, which are selective for the CRF₂ receptor over the CRF₁ receptor (antisauvagine-30 [51] and astressin₂-B [49]).

The pharmacological profile of the CRF₂ receptor has been investigated using the cloned receptor expressed in tissue culture cells. Agonist ligands UCN 1, UCN 2, UCN 3 and sauvagine stimulate cAMP accumulation via mammalian CRF₂ receptors with low nanomolar to sub-nanomolar potency, whereas CRF is less potent [1], [7], [15], [20], [32], [35], [42]. In radioligand binding assays, affinities of approximately 1 nM or below have been reported for UCN 1, UCN 2, sauvagine and the antagonists astressin, antisauvagine-30 and astressin₂-B. UCN 3 and r/hCRF bind less potently (2–14 and 12–190 nM, respectively, and oCRF binds weakly (>100 nM) [1], [7], [15], [20], [32], [35], [42], [44], [49], [51]. Ligands have been identified as selective for the CRF₂ receptor by comparison with affinity for the CRF₁ receptor (UCN 2, UCN 3, antisauvagine-30 and astressin₂-B). Conversely, oCRF is selective for the CRF₁ receptor [13], [15], [20], [52]. The three CRF₂ receptor splice variants exhibit a similar pharmacological profile [1], [32], [35], [42]. Receptor regions involved in binding ligand binding have been identified as the large extracellular domain [13], [44], the juxtamembrane domain [14] and, within this domain, the second extracellular loop [13]. It is probable that the ligand binding orientation is similar to the two-domain mechanism identified for the CRF₁ receptor, in which the C-terminal portion of the ligand binds the extracellular N-terminal domain of the receptor, and the N-terminal portion of the ligand binds the receptor's juxtamembrane domain (reviewed in Refs. [4], [23], [47]).

While the CRF₂ receptor's physiological functions are being rapidly identified, current knowledge of CRF₂ receptor pharmacology is limited. Peptide ligand binding has not been evaluated for endogenously expressed CRF₂ receptors. This evaluation is important because receptor behavior in transfected cells can be affected by the non-native cellular environment. In addition, profiling the endogenously expressed receptor is important in investigating the physiological roles of the CRF₂ receptor, since in these investigations it is necessary to demonstrate a CRF₂ receptor profile in the system of interest. The ligand binding mechanisms of the receptor also require further investigation. Ligand affinity for the CRF₂ receptor is affected by receptor G-protein coupling [1], [13], [20], [50], but the magnitude of this effect has not been quantified. Little is known of the molecular basis of CRF₂/CRF₁ receptor selectivity for recently identified selective agonists (UCN 2, UCN 3) and antagonists (antisauvagine-30 and astressin₂-B). Identifying the receptor domains responsible for selectivity will be important for the further development of receptor selective ligands to probe CRF₂ receptor distribution and function. In this study we have addressed these questions using radioligand binding and functional experiments for the receptor expressed endogenously, for different conformational states of the receptor, and for chimeric CRF₂/CRF₁ receptors.