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Investigating the structure and function of the ECL2 domain of the CGRP receptor The extracellular loops (ECLs) of GPCRs are required for ligand binding and receptor activation. ECL2 is of particular importance in this process (1). A novel modeling approach has been developed, which uses the plant GPCR, GCR1, to bridge the difference between family A and B primary sequences to create a CGRP receptor model (2). The CGRP receptor is a family B GPCR with potent vasodilatory effects combined with an inflammatory and nociceptive response. Using a combined alanine substitution mutagenesis and computer modeling approach, we have previously identified a role for the ECL2 domain of the CGRP receptor in both ligand-binding and activation (3). This latest work has used site directed mutagenesis to create a comprehensive set of 36 new amino acid substitutions of 15 ECL2 residues, previously identified as having functional importance, to further investigate these positions. The effect of the ECL2 mutagenesis on cAMP signaling compared with wild type (WT) receptor was investigated. WT and mutant receptors were transfected into mammalian Cos7 cells and dose dependently stimulated with CGRP (10-12 to 10-6M + basal). The cAMP second messenger response was measured using a TR-FRET based cAMP assay (PerkinElmer). A sigmoidal dose response curve was fitted to the data using GraphPad Prism software. The mutagenesis data was normalised to WT. Experimental replicates were between 3-5 and the significance was obtained through paired t-test. *p < 0.05, ** p < 0.01, ***p < 0.001. The log difference in EC50 values between WT receptor and the alanine/further substitution mutant receptor was taken for each biological replicate to normalize for the difference in WT EC50 values observed for each experiment. The mean ± SEM was calculated. The log difference in EC50 values between the alanine substitution-WT and further substitution-WT was compared using an unpaired t-test to statistically evaluate full/partial/no recovery with the new mutations. Experimental repeats were between 3-5. +p < 0.05, ++ p < 0.01, +++p <0.001, ++++p < 0.0001. Selected results from this analysis are presented in the table below. Key observations include only a partial recovery with the conserved R274K mutation. W283 of the conserved family B CW motif showed partial recovery with an F substitution but full recovery with H.
1. Wheatley, M., et al. (2012) Br J Pharmacol 165, 1688-1703 2. Vohra, S., et al. (2013) J R Soc Interface 10, 20120846 3. Woolley, M. J., et al. (2013) J R Soc Interface 10, 20130589
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