Cannabinoid receptor-G protein interactions: G(alphai1)-bound structures of IC3 and a mutant with altered G protein specificity

Abstract

The structure of the C-terminal region of the third cytoplasmic loop (IC3) of the cannabinoid receptor one (CB1) bound to G(alphai1) has been determined using transferred nuclear Overhauser effects (NOEs). The wild-type IC3 sequence is helical when associated with G(alphai1). In contrast, a peptide containing the amino-acid inversion, Ala(341)-Leu(342) adopts a single turn. These findings correlate with the attenuated G(i) association of CB1 with the Ala(341)-Leu(342) mutation previously observed in vivo and the diminished stimulation of G(alphai1) GTPase activity by the corresponding peptide demonstrated in vitro here. These results, the first to report the structure of a GPCR domain while associated with G protein, imply the C-terminus of CB1 IC3, a region with high-sequence conservation among G-protein coupled receptors, must be helical for efficient coupling and activation of the G(i) protein.

Fig. 1.

GTPase activity of G_αi1. The enzyme assays were conducted as described in Materials and Methods. Each data point represents an average of triplicate assays +/− SE. (A) G_αi1 GTPase activity in the absence and presence of 1 mM peptides and 200 μM GTP. (B) The dependence of G_αi1 GTPase activity on the concentration of the wild-type IC3 peptide (filled circles) and the Ala³⁴¹-Leu³⁴² variant (open circles). (C) The dependence of the peptide-stimulated G_αi1 GTPase activity on GTP concentration. The concentration of the wild-type IC3 peptide (filled circles) and the Ala³⁴¹-Leu³⁴² variant (open circles) was 1 mM.

Fig. 2.

Superposition of the ensemble of 20 structures from the DG calculations: (a) WT-IC3 and (b) AL-IC3. The heavy backbone atoms were used in the superposition.

Fig. 3.

Structure of (a) WT-IC3 and (b) AL-IC3 after IRMA refinement. The Connolly surface representation of the molecules are shown color coded by hydrophobicity (red represents hydrophobic, blue represents hydrophilic).