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Figure 4 | BMC Pharmacology

Figure 4

From: Identification of specific calcitonin-like receptor residues important for calcitonin gene-related peptide high affinity binding

Figure 4

cAMP production of activated wild type and CLR mutants using endogenous or truncated CGRP ligands. Increasing amounts of (A) CGRP, (B) CGRP(1–36) or (C) CGRP(1–19) were used to stimulate confluent HEK293T-RAMP1 cells that had been transiently transfected, individually with wild type (■), L24A (), L34A () or L24A,L34A (x) CLR mutations. (B) Other groups of HEK-RAMP1 cells transiently transfected, individually with wild type (□), L24A (), L34A (+) or L24A,L34A () CLR mutations were treated with 1μM of the CGRP receptor antagonist, CGRP(8–37), for 30 min prior to the addition of 10μM CGRP(1–36). After 30 min all cells were lysed and the amount of cAMP generated was quantified using a radioimmunoassay according to the manufactures protocol (Amersham). Non-linear regression analysis was used to best fit a sigmoidal curve from the data points of each individual experiment. From this best fit curve a concentration of peptide agonist that produced 50% of the maximal cAMP response (EC50) was estimated for each CLR. The calculated EC50 values of all peptides to increase cAMP in HEK293T-RAMP1 cells transiently transfected with L24A, L34A or L24A,L34A CLR mutations were no different from values calculated for the wild type receptor and are displayed in table 2. The data presented represents the mean ± S.E. for n = 3 experiments performed in duplicate.

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