Shown above (A) is the alignment of amino acid sequences of proposed TM4 segments of human family B GPCRs, and of the rabbit calcitonin receptor (rCTR). Highlighted are hCTR residues, Arg236, Val250, and Thr253, which are the sites of mutagenesis in the current study. Shown below (B) is the predicted helical wheel organization of TM3, TM4, and TM5 of hCTR receptor (based on the GLP1 receptor model by Donnelly [35]). Asterisks depict the TM4 residues that are predicted to be exposed to the surrounding lipid in the membrane bilayer.