Regulation of G(i) by the CB1 cannabinoid receptor c-terminal juxtamembrane region: Structural requirements determined by peptide analysis

A CB1 cannabinoid receptor peptide fragment from the C-terminal juxtamembra ne region autonomously inhibits adenylyl cyclase activity in a neuroblastom a membrane preparation. The cannabinoid receptor antagonist, SR141716A, fai led to block the response. The peptide was able to evoke the response in me mbranes from Chinese hamster ovary (CHO) cells that do not express the CB1 receptor. These studies are consistent with a direct activation of G(i) by the peptide. To test the importance of a BXBXXB sequence, Lys(403) was acet ylated, resulting in a peptide having similar affinity but reduced efficacy . N-Terminal truncation of Arg(401) resulted in a 6-fold loss of affinity, which was not further reduced by sequential truncation of up to the first s even amino acids, four of which are charged. N-Terminal-truncated peptides exhibited maximal activity, suggesting that G(i) activation can be conferre d by the remaining amino acids. Truncation of the C-terminal Glu(417) or su bstitution of Glu(417) by a Leu or of Arg(401) by a Norleucine reduced acti vity at 100 mu M. The C-terminal juxtamembrane peptide was constrained to a loop peptide by placement of Cys residues at both terminals and disulfide coupling. This modification reduced the affinity 3-fold but yielded near-ma ximal efficacy. Blocking the Cys termini resulted in a loss of efficacy. Ci rcular dichroism spectropolarimetry revealed that all C-terminal juxtamembr ane peptide analogues exist in a random coil conformation in an aqueous env ironment. A hydrophobic environment (trifluoroethanol) failed to induce alp ha-helix formation in the C-terminal juxtamembrane peptide but did so in le ss active peptides. The anionic detergent sodium dodecyl sulfate induced al pha-helix formation in all analogues except the loop peptide, where it indu ces a left-handed PLT conformation. It is concluded that alpha-helix format ion is not required for G(i) activation.