A peptide comprising the juxtamembrane C-terminal intracellular loop 4 (IL4) of the CB1 cannabinoid receptor possesses three serine residues (Ser402, Ser411, and Ser415). We report the effect of Ser phosphorylation on the CB1 IL4 peptide conformation and cellular signaling functions using nuclear magnetic resonance spectroscopy, circular dichroism (CD), G protein activation, and cyclic adenosine monophosphate (cAMP) production. Phosphorylation at Ser residues induced helical structure in different environments. Helical content varies in the order of IL4p-Ser411 > IL4pSer415 > IL4 > IL4pSer402. The efficacy of phosphorylated IL4 peptides in activating Go and Gi3 ([35S]GTPγS binding) and inhibiting cAMP accumulation in N18TG2 cells was correlated with helicity changes. Bradykinin treatment, which activates protein kinase C (PKC), augmented CB1-mediated inhibition of cAMP accumulation, and this was reversed by a PKC inhibitor. We conclude that phosphorylation-dependent alterations of helicity of CB1 IL4 peptides can augment G protein signaling.
- G protein-coupled receptors (GPCR)
- protein kinase C (PKC)