Residue 17 of Sauvagine Cross-links to the First Transmembrane Domain of Corticotropin-releasing Factor Receptor 1 (CRFR1)

Abstract

Corticotropin-releasing factor (CRF), 2 the main regulator of the hypothalamic pituitary adrenal axis (1), binds to specific G protein-coupled receptors CRFR1 (2, 3) and CRFR2 (4 -7) cloned from several mammalian and nonmammalian species. A third CRF receptor, CRFR3, was found only in fish (8). Several CRF-like peptides were shown to interact with the CRF receptors with high affinity: sauvagine (SVG) characterized from the frog skin (9), urotensin I from the urophysis of fish (10), and urocortin I (UCN1) from the mammalian brain (11). Later, UCN2 and UCN3 (or stresscopins) (12-14) where characterized as CRFR2-specific ligands with little or no affinity to CRFR1.The CRF receptors, which belong to group B of the G protein-coupled receptor superfamily, have six conserved cysteine residues and several N-linked glycosylations in their N-terminal extracellular domains. The extracellular cysteine residues form disulfide bridges that stabilize the structure of the N terminus (15, 16). The N-linked glycosylation moieties are important for processing through the Golgi system and cell surface expression (17). Activation of the cloned CRF receptors stimulates adenylate cyclase (4 -7), phospholipase C (18), and mitogen-activated protein kinases (MAPKs) (19,20).Chemical and/or photoaffinity cross-linking have been extensively used for characterization of ligand-receptor interaction within group B of the G protein-coupled receptor superfamily (21-25), including the CRF receptors (26,27). Recently, the photosensitive amino acid analog p-benzoylphenylalanine (Bpa) was used to substitute several residues within the UCN1 sequence, and photoaffinity cross-linking was used to map the interaction site; the data showed that the very N-terminal ligand residues (1-11) that are responsible for receptor activation are in a close proximity to the second extracellular loop within the juxtamembrane (J-) domain of CRFR1 (26). These data contradict the proposed model derived from the interaction of the free receptor N terminus with the ligand (28 -31). We have previously used an oxidation-resistant SVG analog, [Tyr 0 , Gln 1 , Leu 17 ]SVG (YQL-SVG), which binds to CRFR1 and CRFR2 with high affinity and which cross-links to the CRF receptors with a high efficiency (32), to map the disuccinimidyl suberate-cross-linked residues, and identified covalent crosslinking between Lys 16 of SVG and Lys 257 of CRFR1 in the second extracellular loop (27). In this paper we have used a Bpasubstituted SVG analog, [Tyr 0 , Gln 1 , Bpa 17 ]SVG (YQB-SVG), which has high affinity for CRFR1, and mapped the Bpa 17 crosslinking site to His 117 of CRFR1 and thus established that position 17 of the radioligand lies within 9 Å of position 117 located in the first transmembrane-spanning domain (TM1) of CRFR1.

The “homeostasis hormone” and its CRF1 receptor. From structure to function

“…In particular, upon sauvagine binding to CRF1, residues 17 and 16 were shown to be located in close proximity to residue 117 in the extracellular portion of the TM1 and residue 257 in the second extracellular loop (EL2) of the receptor, respectively. 94,95 The latter finding is in agreement with the results of an alanine mutagenesis study, which has suggested that Trp259 and Phe260 in the EL2 of CRF1 receptor most likely interact with ligands, and specifically with the amino-terminal residues 8-10 of SVG and the corresponding ones of CRF. 96 Furthermore, this study has proposed that the interaction between the amino-terminal region of CRF family peptides and Trp259 and Phe260 of CRF1 seems to be critical for receptor activation and the subsequent appearance of a biological effect.…”

Photo‐Cross‐Linkers Incorporated into G‐Protein‐Coupled Receptors in Mammalian Cells: A Ligand Comparison

“…L42 belongs to a portion of the NTD not involved in ligand binding and was chosen as negative control, while Y73 is located in a loop domain predicted to come into close proximity to the bound ligand [10] (Figure 1). Y116 and Y270 are close to crosslinking sites identified using photoactivatable derivatives of Svg [12] and Ucn 1. [13] To ensure that the mutant receptors correctly interact with the peptide agonists, we measured the stimulated intracellular accumulation of cyclic AMP (data in SI).…”

Exploring the Binding Site Crevice of a Family B G Protein-Coupled Receptor, the Type 1 Corticotropin Releasing Factor Receptor

“…These receptors also have a large extracellular aminoterminal region (N-region) that has been structurally characterized in NMR and crystallographic studies (Grace et al, 2007;Pioszak et al, 2008;Underwood et al, 2010). The N-region and the extracellular loops of family B GPCRs have been shown to play an important role in peptide binding (Holtmann et al, 1996;Liaw et al, 1997;Perrin et al, 1998;Dautzenberg et al, 1999;Unson et al, 2002;Kraetke et al, 2005a;Grace et al, 2007;Assil-Kishawi et al, 2008;Pioszak et al, 2008;Gkountelias et al, 2009).…”