Alanine series of ovine corticotropin releasing factor (oCRF): a structure-activity relationship study

Abstract

Previous structure-activity relationship studies of CRF have shown that residues 1-4 were not necessary for receptor binding or transduction, that residues 4-8 were important for activation, and that residues 12-41 were mostly responsible for binding. Finally it was proposed that CRF assumed an alpha-helical structure when interacting with its receptor. By systematic substitution of each residue (except residues 1-4) in ovine CRF (oCRF) by Ala, we have investigated the role played by individual side chains in receptor recognition and activation. Out of 33 analogues (synthesized using SPPS on an MBHA resin, purified by RPHPLC and characterized by amino acid and mass spectral analyses), a significant loss of biological potency (less than 1% potency of native) was observed for 6 analogues ([Ala6], [Ala8], [Ala10], [Ala12], [Ala14], and [Ala38]); 12 analogues had biological potencies ranging from 1% to 60% and ranked as follows: [Ala35] less than [Ala16] less than [Ala9] less than [Ala19] less than [Ala15] less than [Ala13] less than [Ala7] less than [Ala23] less than [Ala11] less than or equal to [Ala21] less than [Ala27] less than or equal to [Ala18]; 8 analogues were found to be equipotent (greater than 60% and less than 150%) ([Ala5], [Ala17], [Ala26], [Ala29], [Ala30], [Ala34], [Ala36], and [Ala37]; and 7 analogues were found to be approximately 2-5 times more potent than native oCRF ([Ala25] = [Ala40] less than or equal to [Ala39] less than or equal to [Ala33] less than [Ala20] less than [Ala22] less than [Ala32], in an in vitro pituitary cell culture assay. In summary, the Ala substitutions which showed the greatest loss of potency (less than 1% of native oCRF) were those replacing hydrophobic residues while those showing the greatest increase in potency were replacing hydrophilic residues. Of the 22 Ala-containing analogues in the C-terminal half of the molecule, 17 analogues have equal or greater potencies than native oCRF. Substitution of Ala in the N-terminal region (residues 5-19) on the other hand is generally detrimental to biological activity. These results suggest that the side chains of residues 5-19 are very important for receptor binding and activation while, in the C-terminal region, the amino acid side chains may be more responsible for structural conservation than for functional expression.

Biomimetic Screening of Class-B G Protein-Coupled Receptors

“…, and Thr 10 abolished activity. Consistent with our truncation study (section 3.1.4) and with prior studies on full length CRH (Beyermann et al 1996;Kornreich et al 1992), the side chains of these three residues seem to be essential for CRHR 1 activation. Ser 4 could be advantageously substituted for several amino acids.…”

“…UCN is closely related to the CRH hormone and a highly potent nonselective CRHR 1 and CRHR 2 agonist (see Introduction, figure 3 and table 2). Based on several structure-activity-relationship (SAR) studies of the full length CRH peptide hormone, we chose to first investigate whether the fragment 1-15 of the UCN hormone could provide the required amino acid residue sequence that leads to CRHR 1 activation (Beyermann et al 2000;Kornreich et al 1992). Having proven that a "clicked" conjugate can act as high potency agonist, we went on to determine the minimal N-terminal peptide sequence that is necessary to fully activate the CRHR 1 .…”

Biomimetic screening of class B G protein-coupled receptors

“…Replacement of polar residues Ser 6 , Asp 8 , and Thr 10 abolished activity. Consistent with our truncation study (section 3.1.4) and with prior studies on full length CRH (Beyermann et al 1996;Kornreich et al 1992), the side chains of these three residues seem to be essential for CRHR 1 activation. Ser 4 could be advantageously substituted for several amino acids.…”

“…In particular, Rivier et al have performed alanine and D-amino acid scans of the whole CRH sequence (Kornreich et al 1992;Rivier et al 1993). As CRH and UCN share a high degree of homology in the activation segment (e.g., the fragments 4-7 in UCN and the corresponding 5-8 in CRH are only slightly different, the Ser 6 is conserved while Leu residues are exchanged by Ile residues and vice versa; the UCN 8-15 fragment is completely conserved in CRH 9-16 ; Table 2 (Beyermann et al 1996).…”

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

“…In contrast, there is great sequence diversity within the C-terminal region . Substitutions of Arg-35 or Leu-38 in oCRF by alanine (18), conversion of the C-terminal carboxamide to a carboxyl group or truncation of the C-terminal dipeptide from oCRF, however, reduced biopotency dramatically (14), indicating an essential binding site to be located at the extreme of the C terminus. The existence of two receptor binding sites in peptide ligands of class 2 GPCRs was also suggested by studies using chimeric receptors and peptide ligands, but nothing has been described concerning the structural organization of the ligands (22,23).…”