Impact of N-Terminal Domains for Corticotropin-Releasing Factor (CRF) Receptor−Ligand Interactions

Abstract

The large extracellular N-terminal domains (NTs) of class B G protein-coupled receptors serve as major ligand binding sites. However, little is known about the ligand requirements for interactions with these receptor domains. Recently, we have shown that the most potent CRF receptor agonist urocortin 1 (Ucn1) has two segregated receptor binding sites Ucn1(1-21) and Ucn1(32-40). For locating the receptor domains interacting with these two sites, we have investigated the binding of appropriate Ucn1 analogues to the receptor N-termini compared to the corresponding full-length receptors. For this purpose receptor NTs of CRF(rat) subtypes 1 and 2(alpha) without their signal sequences were overexpressed in Escherichia coli and folded in vitro. For CRF2(a)-rNT, which bears five cysteine residues (C2-C6), the disulfide arrangement C2-C5 and C4-C6 was found, leaving C3 free. This is consistent with the disulfide pattern of CRF1-rNT, which has six cysteines and in which C1 is paired with C3. Binding studies of N-terminally truncated or C-terminally modified Ucn1 analogues demonstrate that it is the C-terminal part, Ucn1(11-40), that binds to receptor NT, indicating a two-domain binding mechanism for Ucn binding to receptor NT. Since the binding of Ucn1 to the juxtamembrane domain has been shown to be segregated from binding to the receptor N-terminus [Hoare et al. (2004) Biochemistry 43, 3996-4011], a third binding domain should exist, probably comprising residues 8-10 of Ucn, which particularly contribute to a high-affinity binding to full-length receptors but not to receptor NT.

Corticotropin Releasing Hormone - A GPCR Drug Target

“…[3] Included in the class B GPCRs are calcitonin, parathyroid hormone, glucagon-like peptide, and GHRH, some of the applications, current and potential include osteoporosis and Type II diabetes. [16,22] There are three types of CRH receptors that have been identified. CRHR 1 and 2 share 70% homology on the amino acid level and are coded for by separate genes.…”

“…A hypothesis was proposed for the mechanism of binding for class B GPCRs by peptide agonists (such as CRH) involving a two-domain process (Fig (2)). [7,[15][16][17]22] The N-terminus of the receptor initially binds the carboxyl terminus of the peptide; this then initiates rearrangement of the receptor with the insertion of the peptide agonist amino terminus into the transmembrane helix bundle and activation of the receptor ( Fig. (2), Step 1 and insert).…”

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

“…83 Similarly, a soluble form of the N-domain of CRF1 as well as a chimera created by replacing the extracellular region of activin receptor (a single membrane-spanning segment receptor) with the N-domain of CRF1 have been shown to bind to UCN and astressin with affinities that are lower than wild type receptor but still biologically considerable. [84][85][86] Structure-function studies have established that the amino acids, important for ligand binding, in the N-domain of CRF1 are located between residues 43-50 and 76-84 of the receptor. 87,88 The precise interactions of the N-domain of CRF receptors with various peptides have been determined by crystallography and NMR studies using soluble forms of this receptor's segment.…”

“…89 The N-domain of CRF1 receptor also contains three disulfide bridges between Cys30 and Cys54, Cys44 and Cys87 and Cys68 and Cys102, which play a crucial role in maintaining the receptor in its functional conformation. 86,89,91 Reduction of these bonds with DTT or mutation of Cys which participate in these bonds to Ser or Ala significantly decreased CRF binding. 92 The disulfide arrangement in the N-domain of CRF2β is identical to that of the corresponding region of CRF1, but different than that in the N-domain of CRF2α, which has four Cys linked to each other with disulfide bonds and one free Cys.…”

“…92 The disulfide arrangement in the N-domain of CRF2β is identical to that of the corresponding region of CRF1, but different than that in the N-domain of CRF2α, which has four Cys linked to each other with disulfide bonds and one free Cys. 85,86,89,93 In addition to the N-domain of CRF receptors, their extracellular loops as well as the extracellular portions of their TMs have been revealed as important for peptide binding. 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.…”