Structural analysis of the human cannabinoid receptor one carboxyl-terminus identifies two amphipathic helices.
Recent research has implicated the C-terminus of G-protein coupled receptors in key events such as receptor activation and subsequent intracellular sorting, yet obtaining structural information of the entire C-tail has proven a formidable task. Here, a peptide corresponding to the full-length C-tail of the human CB1 receptor (residues 400-472) was expressed in E.coli and purified in a soluble form. Circular dichroism (CD) spectroscopy revealed that the peptide adopts an alpha-helical conformation in negatively charged and zwitterionic detergents (48-51% and 36-38%, respectively), whereas it exhibited the CD signature of unordered structure at low concentration in aqueous solution. Interestingly, 27% helicity was displayed at high peptide concentration suggesting that self-association induces helix formation in the absence of a membrane mimetic. NMR spectroscopy of the doubly labeled ((15)N- and (13)C-) C-terminus in dodecylphosphocholine (DPC) identified two amphipathic alpha-helical domains. The first domain, S401-F412, corresponds to the helix 8 common to G protein-coupled receptors while the second domain, A440-M461, is a newly identified structural motif in the distal region of the carboxyl-terminus of the receptor. Molecular modeling of the C-tail in DPC indicates that both helices lie parallel to the plane of the membrane with their hydrophobic and hydrophilic faces poised for critical interactions.
Ahn
Kwang H
KH
Department of Molecular and Cell Biology, University of Connecticut, Storrs, CT 06269, USA.
Pellegrini
Maria
M
Tsomaia
Natia
N
Yatawara
Achani K
AK
Kendall
Debra A
DA
Mierke
Dale F
DF
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D017433:Protein Structure, Secondary; D043882:Receptors, Cannabinoid / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism
D010455:Peptides; D043882:Receptors, Cannabinoid