Conformational Characterization of Phosphorylated Analogs of a Peptide Mimetic of the Fourth Cytoplasmic Loop of CB1Cannabinoid Receptor

Abstract

Phosphorylation is an important regulatory mechanism in receptor signaling. It has been shown that phosphorylation of the CB1 receptor disrupts modulation of ion channels by the receptor and CB1 cannabinoid receptor intracellular C-terminal tail domain (amino acids 401-417) is critical for G(i/o) protein coupling. Further, the proximal portion of the C-terminus of the cannabinoid CB1 receptor is a primary determinant for G-protein activation. The synthetic peptide fragment of the C-terminal juxtamembrane region (CB1 401-417) referred here as IL4 peptide mimicked the receptor's response of inhibiting adenylate cyclase. In the present study, we have used phosphorylated analogues of IL4 peptide to analyze the effect of phosphorylation on the conformation of the peptide and G-protein activation using NMR Spectroscopy. Unambiguous proton NMR assignments have been carried out with the aid of correlation spectroscopy (DQF-COSY and TOCSY) experiments and nuclear Overhauser effect spectroscopy (NOESY and ROESY) experiments. The distance constraints obtained from the NMR data have been used in torsion angle dynamics algorithm for NMR applications (DYANA) to generate a family of structures which have been refined using restrained energy minimization and dynamics. The conformational range of the phosphorylated IL4 peptide revealed by NMR studies has been analyzed in terms of characteristic secondary structural features. The results obtained provide insight into the mechanism by which the peptide activates G-proteins, as a first step in signal transduction.