Book of Abstracts: Albany 2009
June 16-20 2009
© Adenine Press (2008)
Conformation of a Peptide Mimetic of the Fourth Cytoplasmic Loop of the CB1 Cannabinoid Receptor
The CB1 cannabinoid receptor is a G-protein coupled receptor that regulates multiple signal transduction pathways, including inhibition of adenyl cyclase and regulation of ion channels.The intracellular surface of the CB1 receptor interacts directly with selective G-proteins. The juxtamembrane C-terminal region is critical for G-protein and signal transduction regulation. Thus, the determination of structural changes in this domain can provide insight into the mechanisms for efficacy in signal transduction. A synthetic peptide fragment of the C-terminal region of CB1 (residues 401-417) has been shown to activate Go and Gi proteins in a pertussis toxin-sensitive manner. This receptor domain is expected to be palmitoylated at cysteine 416, and the structure imposed by this membrane anchor is believed to be influential in the interactions between receptor and G-protein. Circular dichroism (CD) studies of the peptide in water, sodium phosphate buffer, and methanol are characteristic of random coil structures, whereas the addition of sodium dodecyl sulfate (SDS) or dipalmitoylphosphatidylglycerol induces helical structure. The addition of trifluoroethanol (TFE) to provide a hydrophobic environment does not induce helical structure in this peptide. Structural investigations using 2D-NMR in water show extended coil conformation and in SDS micelles show the formation of helical structure. The distance constraints from the NMR data have been used in a torsion angle dynamics algorithm and molecular dynamics simulations to produce a model of the peptide as a helix with cationic clusters largely oriented toward the cytoplasm. This structure appears to be modified by the environment, such as might be imposed by protein-protein interactions.
Phosphorylation is an important regulatory mechanism in signal transduction. Structural investigations were made on the CB1 peptide fragment with each serine phosphorylated. CD spectrophotometry on the S402-phosphorylated peptide showed the presence of no secondary structure in phosphate buffer and a shift toward helicity in 50% methanol. A solution of SDS induced helicity, but to a lesser degree than TFE, which began to exhibit helical structure at concentrations as low as 20% TFE and exhibited a significant number of residues in a helical conformation at 90% TFE. Initial NMR data confirm that the degree of helical structure increases as the concentration of TFE increases. 2D-NMR data show no ordered structure in water or low concentrations of TFE. The induction of helicity in TFE for the phosphorylated peptide but not the native fragment suggests a conformational shift in this region upon phosphorylation that may play a role in signal transduction.
1School of Life Sciences