A Role for a Helical Connector between Two Receptor Binding Sites of a Long-chain Peptide Hormone

Abstract

The conformational freedom of single-chain peptide hormones, such as the 41-amino acid hormone corticotropin releasing factor (CRF), is a major obstacle to the determination of their biologically relevant conformation, and thus hampers insights into the mechanism of ligand-receptor interaction. Since N-and C-terminal truncations of CRF lead to loss of biological activity, it has been thought that almost the entire peptide is essential for receptor activation. Here we show the existence of two segregated receptor binding sites at the N and C termini of CRF, connection of which is essential for receptor binding and activation. Connection of the two binding sites by highly flexible ⑀-aminocaproic acid residues resulted in CRF analogues that remained full, although weak agonists (EC 50 : 100 -300 nM) independent of linker length. Connection of the two sites by an appropriate helical peptide led to a very potent analogue, which adopted, in contrast to CRF itself, a stable, monomer conformation in aqueous solution. Analogues in which the two sites were connected by helical linkers of different lengths were potent agonists; their significantly different biopotencies (EC 50 : 0.6 -50 nM), however, suggest the relative orientation between the two binding sites rather than the maintenance of a distinct distance between them to be essential for a high potency.

Biomimetic screening of class B G protein-coupled receptors

“…This is consistent with the results of Beyermann et al (Beyermann et al 2000). Taken together, these results allow us to propose a three-step "low resolution" mechanism for the binding events of our UCN 4-15 "clicked" conjugates.…”

Synthetic Multivalent Ligands as Probes of Signal Transduction

“…testosterone) release during the stress response. [234] A series of dimers was generated, such that each component possesses REs in different orientations; the orientation of the REs was controlled by using a helical and rigid linkers. The addition of these dimers to cells expressing the CRFR-1 resulted in testosterone release.…”

Biomimetic Screening of Class-B G Protein-Coupled Receptors

“…Stepwise, the C-terminal part of the hormone binds the CRHR 1 extracellular domain (CRHR 1 -ECD1) with high-affinity, an interaction that directs the N-terminal part of the hormone to interact with the CRHR 1 transmembrane domain (CRHR 1 -TMD) leading to G-protein signaling. In particular, the middle domain of the CRH hormone, which connects the two functional sites of the peptide hormone, was successfully replaced by a helical "connector", thereby reconstituting a potent peptide agonist (Beyermann et al 2000). Based on this work, we developed a peptide-peptide conjugation strategy to chemically probe the molecular interactions of CRH-like peptide ligands with their cognate receptor.…”

“…In particular, we aimed to address the following questions: (Beyermann et al 2000). CRHR 1 peptide ligands bind and activate their cognate class B GPCR thanks to a low resolution "two domain" mechanism.…”