Figure 4. Non-conservative amino acid substitutions may contribute to constitutive activity in SmGAR.

(A) A homology model of SmGAR (red) was superimposed onto the crystal structure of the rat M3 muscarinic receptor (4daj) (blue). The SmGAR model shows a typical topology of Family A GPCRs, including the canonical 7 transmembrane (TM) domains. Residues of the predicted “ionic lock” of Family A GPCRs are shown at the cytosolic ends of TM 3 and TM 6. Residue R^3.50 corresponds to Arg248^3.50 in SmGAR and Arg165^3.50 in the rat M3 receptor. E^6.30 refers to Glu485^6.30 of the rat M3 receptor and A^6.30 is the corresponding alanine in SmGAR (Ala848^6.30). (B) Structural alignment of vertebrate and invertebrate muscarinic receptors showing a portion of TM 3 and TM6. The numbers describe the relative positions of amino acids of interest in each TM helix, according to the Ballasteros and Weinstein system [33], as described in the text. Mammalian muscarinic receptors are identified according to subtype, M1–M5; Invertebrate receptors are listed as GAR (G protein-coupled acetylcholine receptor) or mAChR (muscarinic acetylcholine receptor). Species abbreviations are as described in Fig. 1 and accession numbers are provided in Table S1.