title: Structure of the third intracellular loop of the human cannabinoid 1 receptor.
abstract: The third cytoplasmic loop (IC3) is a determinant in the dynamic life cycle of G protein-coupled receptors, including the activation, internalization, desensitization, and resensitization processes. Here, we characterize the structural features of the IC3 of the cannabinoid 1 receptor (CB1) in micelle solution using heteronuclear, (1)H,(15)N-high-resolution NMR methods. The IC3 construct was designed to contain one-third of each of the transmembrane helices (TMs 5 and 6) to tether the protein to the hydrophobic portion of the micelle. Indeed, the NMR analysis illustrates prominent alpha-helices at the N-terminus (G1-R10) and C-terminus (Q37-T47) of the IC3 receptor domain, corresponding to the cytoplasmic termini of TM5 and TM6. The structural features of the central portion of the IC3 consist of a small alpha-helix, adjacent to the terminus of TM5. The remainder is mostly unstructured as indicated by the NMR-based observables (NOEs and chemical shifts). Despite the lack of secondary structure, the hydrophobic triplet of isoleucine residues in the center of the IC3 is found in molecular dynamics simulations to associate with the lipid environment, producing two smaller loops out of the IC3. Previous studies examining mastoparan and related peptides and their ability to activate G proteins have concluded an alpha-helix is required for efficient binding and activation. Our structural results for the IC3 of CB1 would then suggest that in the intact receptor the G protein is activated by the alpha-helices of the cytoplasmic ends of TM5 or TM6 and not the unstructured central region of the IC3.
authors.affiliation: Department of Molecular Pharmacology, Division of Biology and Medicine, Brown University, Providence, Rhode Island 02912, USA.
authors.forename: Amy L
authors.initials: AL
authors.lastname: Ulfers
authors.forename: Jonathan L
authors.initials: JL
authors.lastname: McMurry
authors.forename: Debra A
authors.initials: DA
authors.lastname: Kendall
authors.forename: Dale F
authors.initials: DF
authors.lastname: Mierke
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D006801:Humans; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D043882:Receptors, Cannabinoid; D011955:Receptors, Drug / Q000737:chemistry* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012243:Rhodopsin / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013917:Thrombin / Q000378:metabolism
chemical_list: D043882:Receptors, Cannabinoid; D011955:Receptors, Drug; D011993:Recombinant Fusion Proteins; D012243:Rhodopsin; D013917:Thrombin