Conformational states of the corticotropin releasing factor 1 (CRF1) receptor: detection, and pharmacological evaluation by peptide ligands
Introduction
Corticotropin releasing factor (CRF) is a 41 amino acid peptide that regulates the hypothalamic–adrenal–piuitary axis and central signaling in the physiological response to stress [40], [45]. These actions of CRF are known to be mediated by the CRF1 receptor [2], [4], [48], a secretin-family G-protein-coupled receptor (GPCR). CRF and the CRF1 receptor form part of a group of related signaling molecules, which include the CRF2 receptor [30], and the peptides urocortin I (UCN I) [47], UCN II [24], [39], UCN III [24], [29] and the amphibian peptide sauvagine [33] (Fig. 1). Anatomical, behavioral and genetic studies implicate alteration of the CRF system in anxiety and depression, potentially involving both CRF1 and CRF2 receptors [10], [16], [23].
The pharmacological profile of the CRF1 receptor has been extensively investigated, owing to its important physiological role and its potential as a drug target. The ligand-activated receptor is coupled to stimulation of cAMP accumulation, presumably via stimulation of Gs G-proteins. Potent activation of the cloned CRF1 receptor has been demonstrated for CRF and UCN I from various species, and for sauvagine [2], [4], [7], [13], [35], [43], [48], [50]. These peptides also potently activate the endogenously expressed receptor, demonstrated by measuring cAMP accumulation in pituitary, brain and spleen tissue, and release of adrenocorticotropic hormone from pituitary cells (reviewed in [15]). The cloned CRF1 receptor is not activated by physiological concentrations of UCN II [24], [39] or UCN III [24], [29].
The ligand binding properties of the CRF1 receptor have been investigated predominantly by measuring displacement of agonist radioligand binding to cell membranes prepared from tissues [9], [14], [15], [34], [36], [49], or membranes prepared from cells expressing the cloned receptor [4], [7], [43], [48]. Binding of agonist radioligands is inhibited with high affinity by the agonists CRF, UCN I and sauvagine (<1–10 nM), and by the antagonists astressin [32] (1 nM) and α-helical CRF [42] (30 nM). Binding is poorly inhibited by UCN II (Ki>1 μM) [1], [39] and UCN III (Ki>100 nM) [29], and weakly inhibited by the CRF2-selective antagonists antisauvagine-30 (150 nM) [44] and astressin2-B (>500 nM) [41]. Radiolabeled sauvagine and CRF binding to the CRF1 receptor is reduced by guanine nucleotides, so it is likely that this pharmacological profile is that of the G-protein-coupled state of the receptor (RG) [7], [9], [14], [34], [36], [37], [43], [49].
The pharmacological profile and relative abundance of other CRF1 receptor states are largely unknown. In common with other GPCRs, the CRF1 receptor also exists in a G-protein-uncoupled state (R) bound by the labeled antagonist []astressin [7], [37], [43]. Ligand affinity for this state has not been unambiguously defined. Defining the R state pharmacologically is important, because this state might represent the majority of CRF1 receptors [7], [37], [43] but is rarely considered in studies of CRF1 receptor expression, localization, and binding mechanism. In addition, non-peptide antagonist activity differs markedly between RG and R states; the compounds strongly inhibit peptide binding to the former but only weakly inhibit binding to the latter [22]. Studies of detergent-solubilized CRF1 receptor suggest the presence of a third state of the receptor bound with high affinity by radiolabeled agonist but insensitive to guanine nucleotides [19]. It is not known if this state is an artifact of solubilization or is present in membrane preparations.
The aims of this study were to identify the conformational states of the cloned CRF1 receptor expressed in mammalian cells and the receptor endogenously expressed in rat cerebellum, to pharmacologically define these states with a large array of ligands, and to quantify the abundance of these states.
Section snippets
Materials
Peptides were synthesized by solid phase methodology on a Beckman Coulter 990 peptide synthesizer (Fullerton, CA) using t-Boc-protected amino acids. The assembled peptide was deprotected with hydrogen fluoride. The crude peptide product was purified by preparative HPLC and the purity of the final product was assessed by analytical HPLC and mass spectrometric analysis using an ion-spray source. Peptides were quantified by weight; the net peptide content was greater than 90% as assessed by
Modulation of radioligand binding to L-CRF1 cell membranes by GTPγS
The CRF1 receptor was stably expressed in Ltk− cells, and binding of radioligands to membranes prepared from these cells was assessed. The total binding:non-specific binding ratio for []sauvagine (9–23) was considerably greater than the ratio for []oCRF (2.6–3.5). Binding of the antagonist radioligand []astressin was also readily detectable (total binding:non-specific binding ratio of 7–11). No specific binding of these radioligands was detectable in membranes from Ltk− cells that
Discussion
The aim of this study was to identify and pharmacologically characterize conformational states of the CRF1 receptor. The cloned receptor expressed in Ltk− cells was initially used, to allow examination of the receptor in isolation. Subsequently the presence of receptor states and their pharmacological profile were evaluated for the receptor expressed endogenously in rat cerebellum. The principle findings of this study are: (1) Three affinity states of the cloned receptor were identified. [
Acknowledgements
We gratefully acknowledge Xin-Jin Liu for the preparation of [125I]astressin.
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