Structure-activity relationships in calcitonin gene-related peptide: Cyclic AMP response in a preosteoblast cell line (ks-4)

Abstract

Synthetic analogs of chicken and human CGRP were used to define structure-function relationships in a murine osteoblast precursor cell line, KS-4, that exhibits a substantial cyclic AMP response to CGRP but no response to calcitonin. Human CGRP had 40% of the activity of chicken CGRP, but [Asp-14]-human CGRP was equipotent with chicken CGRP. Similar observations were made previously for calcium- and phosphate-lowering effects. Des-1-Ala,deamino CGRP compounds of both human and chicken origin were three-fold more potent than the respective native CGRPs. The [4-F-Phe)-37]-chicken CGRP analog had a four-fold enhanced activity. In all of the 13 analogs either truncated amino-terminally or missing the (2-7)-disulfide bridge, biologic activity was greatly reduced (0.1% of chicken CGRP), as it was in chicken CGRP-(1-36)-OH, which lacks the C-terminal amino acid. The same analog has been shown to retain a hypocalcemic effect, which is most likely mediated through calcitonin receptors. In confirmation of their antagonistic effect reported in liver, analogs that lack the N-terminal ring structure exhibited significant antagonistic activity. The data illustrate the specificity of CGRP receptors in promoting cyclic AMP formation in osteoblast-like cells. Such CGRP analogs will be useful in investigating structure-function relationships of CGRP.

Structural comparison of alanine‐substituted analogues of the calcitonin gene‐related peptide 8‐37 Importance of the C‐terminal segment for antagonistic activity

“…When the structure of this segment is conserved, as is the case for [Ala'7]-hCGRP 8-37 and [AlaZo]-hCGRP 8-37, the antagonistic activity of the peptide for CGRPl receptors is conserved (Table 1 ). They can also be paralleled with the loss of activity observed for hCGRP in CGRPl receptors when the terminal phenylalanine residue was removed or chemically modified (22)(23)(24). The existence of hydrogen bonding between the amide carbonyl oxygen of and the amide NH hydrogen of Asn3' appears to be an essential feature for activity.…”

The Activity of Peptides of the Calcitonin Family in Bone

“…Specific binding of CGRP to cells of rat calvaria was demonstrated (186). CGRP treatment increased cAMP levels in the UMR 106-01 rat osteosarcoma cell line and in other osteoblastic cell lines and primary osteoblast cultures (18,148,200). In primary bone cell cultures from neonatal chicken, rat, and mouse calvariae, CGRP increased cAMP levels, whereas calcitonin had no effect on these cells (149).…”

Structure–activity relationships of the N‐terminus of calcitonin gene‐related peptide: key roles of alanine‐5 and threonine‐6 in receptor activation

“…There has been some work on the structure-activity relationship for the C-terminus of CGRP acting on the CGRP receptor (Conner et al, 2002;Lang et al, 2006), but much less is known about the N-terminus of the peptide when it activates this receptor or the AMY1 receptor . The importance of the disulphide bond has been established (Dennis et al, 1989;Saha et al, 1998), and there are a few studies based mainly on deletions (Thiebaud et al, 1991;Hakala et al, 1994;Heino et al, 1998). It is possible to extend the N-terminus of CGRP, as with [Tyr 0 ]-CGRP, while retaining high affinity (Dennis et al, 1989) although efficacy may be compromised (Poyner et al, 1992).…”