Identification of Amino Acids in the N‐Terminal Domain of Corticotropin‐Releasing Factor Receptor 1 that Are Important Determinants of High‐Affinity Ligand Binding

Abstract

Abstract:The aim of the present study was to identify the N-terminal regions of human corticotropin-releasing factor (CRF) receptor type 1 (hCRF-R1) that are crucial for ligand binding. Mutant receptors were constructed by replacing specific residues in hCRF-R1 with amino acids from the corresponding position in the N-terminal region of the human vasoactive intestinal peptide receptor type 2 (hVIP-R2). In cyclic AMP stimulation and CRF binding assays, it was established that two regions within the N-terminal domain were crucial for the binding of CRF receptor agonists and antagonists: one region mapping to amino acids 43-50 and a second amino acid sequence extending from position 76 to 84 of hCRF-R1. Recently, it was found that the latter sequence plays a very important role in determining the high ligand selectivity of the Xenopus CRF-R1 (xCRF-R1). Replacement of amino acids 76 -84 of hCRF-R1 with residues from the same segment of the hVIP-R2 N terminus markedly reduced the binding affinity of CRF ligands. Mutation of Arg 76 or Asn 81 but not Gly 83 of hCRF-R1 to the corresponding amino acids of xCRF-R1 or hVIP-R2 resulted in 100 -1,000-fold lower affinities for human/rat CRF, rat urocortin, and astressin. These data underline the importance of the N-terminal domain of CRF-R1 in high-affinity ligand binding. Key Words: Corticotropin-releasing factor receptor 1 mutagenesis-Ligand binding-Cyclic AMP-Corticotropinreleasing factor binding site. J. Neurochem. 72, 388 -395 (1999).The 41-amino acid peptide corticotropin-releasing factor (CRF) (for references, see Vale et al., 1981) is the main integrator of the stress response (Dunn and Berridge, 1990;Owens and Nemeroff, 1991). Two major subtypes of the CRF receptor (CRF-R) have been identified in vertebrates as belonging to the superfamily of G protein-coupled receptors (GPCRs): CRF-R type 1 (CRF-R1) and type 2 (CRF-R2). The cDNAs for CRF-R1 and CRF-R2 have been cloned from human Vita et al., 1993;Liaw et al., 1996), rat (Chang et al., 1993;Perrin et al., 1993;Lovenberg et al., 1995), mouse (Vita et al., 1993;Kishimoto et al., 1995;Perrin et al., 1995;Stenzel et al., 1995), chicken (Yu et al., 1996), and amphibian (Dautzenberg et al., 1997) tissues. Two functional splice variants, CRF-R2␣ and CRF-R2␤, have been found for CRF-R2. CRF-R2␣ cDNA encodes a protein of 411-413 amino acids (Lovenberg et al., 1995;Liaw et al., 1996;Dautzenberg et al., 1997), whereas the CRF-R2␤ protein comprises 430 -438 amino acids (Kishimoto et al., 1995;Lovenberg et al., 1995;Perrin et al., 1995;Stenzel et al., 1995;Valdenaire et al., 1997). Both splice variants differ in the N-terminal region and are ϳ70% identical to CRF-R1.High-affinity ligand binding is an important prerequisite for the signal transduction of GPCRs. Small nonpeptidic molecules can bind to the transmembrane domains (TMs) of their receptors (Dixon et al., 1987;Wheatley et al., 1988;Strader et al., 1989), whereas the contribution of extracellular domains (ECs) and TMs has been identified as parts of the binding site of GP...

Molecular cloning and initial characterization of African green monkey (Cercopithecus aethiops) corticotropin releasing factor receptor type 1 (CRF1) from COS-7 cells

“…Although nucleotide substitutions were evenly spread across the mRNA sequence, those resulting in changes in amino acid sequence were predominantly located in the extracellular domain (substrate binding domain). This is in agreement with the known increased susceptibility to mutations changing the amino acid sequence in the N-terminal domain of CRF1 involved in CRF binding (Perrin, Vale, 1999;Wille et al, 1999;Hillhouse, Grammatopoulos, 2006).…”

Human Mast Cells Express Corticotropin-Releasing Hormone (CRH) Receptors and CRH Leads to Selective Secretion of Vascular Endothelial Growth Factor

“…The major ligand-binding determinant of mammalian CRH-R1 has been mapped to its first extracellular domain, coded by exons 1-4 of CRH-R1 (35). Exon 3 contains two regions that are critical for high-affinity ligand binding, and mutations in this region abolish CRH binding (36). CRH-R1␣, 1f, and 1g encode the entire CRHbinding domain; they should, therefore, be the efficient receptor variants that bind and transduce peptide signals into cAMP-mediated pathways.…”

Genetic variation in the corticotrophin-releasing factor receptors: identification of single-nucleotide polymorphisms and association studies with obesity in UK Caucasians

“…Previous investigations of the structurefunction relationship of CRF-R1 have identified the Nterminal domain, the second extracellular domain and junction of the third extracellular domain and fifth transmembrane domain, as regions involved in receptor ligand binding and/or receptor activation. [18][19][20] Although CRF-R1b has been shown to bind CRF with two-fold lower affinity than CRF-R1, the ability of CRF to stimulate adenylate cyclase was reduced 100-fold with CRF-R1b, suggesting that this loop participates in the interaction with G proteins. 21 The physiological role of this splice variant is unclear and the functional significance of Val161Met remains to be determined.…”