Structural analysis of the human cannabinoid receptor one carboxyl‐terminus identifies two amphipathic helices

Abstract

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 α-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 (15N- and 13C-) C-terminus in dodecylphosphocholine (DPC) identified two amphipathic α-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.

Allosteric Modulation of a Cannabinoid G Protein-coupled Receptor

“…In running Modeler on the C terminus, only the unstructured regions were explored; Ala-440 -Met-461 were modeled as an ␣-helix that would be parallel to a lipid bilayer (i.e. the same plane as Helix 8); this is consistent with NMR results that suggest the existence of "Helix 9" in the C terminus (31).…”

Human Cannabinoid 1 GPCR C-Terminal Domain Interacts with Bilayer Phospholipids to Modulate the Structure of its Membrane Environment

“…Our molecular dynamics simulations (27) and SDSL/EPR results (3) further demonstrate that TMH7's intramembrane conformation and relative orientation with respect to H8 are dynamically modulated by its membrane environment. A final characteristic of hCB1(TMH7/H8) germaine to the present work, as first defined by us (4,27) and substantiated by others (30), is the amphipathicity of H8: the relative disposition therein of a hydrophilic cationic cluster contralateral to a hydrophobic "face" of nonpolar residues is likely important for optimal H8 orientation/interaction with both the plasma membrane and the G-protein subunits to which hCB1 couples for information transmission and intracellular signal propagation.…”

“…The relatively limited homology between rhodopsin and hCB1-particularly with respect to their C-terminal domain-precludes direct extrapolation of the rhodopsin structure to hCB1 (25,26). Our and others' investigations on hCB1 higherorder structure indicate that H8 acquires a helical conformation in membrane-mimetic environments with a juxtamembrane orientation parallel to the membrane surface and perpendicular to the TMH7 intramembrane bundle (4,(27)(28)(29)(30). Multiple electrostatic amino-acid interactions underpin TMH7/H8 conformation (4), and interactions between these two hCB1 α-helical segments have been implicated in ligandinduced hCB1 conformational transitioning (3).…”

“…Except for H8, very little direct structural information is available for the C-terminal region of GPCRs, including hCB1 (2,14,30). We previously solved the NMR solution structure of a synthetic peptide 40-mer, hCB1(TMH7/H8), representing the hCB1 C-terminal domain (4) and reported the first application of solid-state NMR, along with SDSL/ EPR, to study the peptide's higher-order structure in defined phospholipid bilayers (3,27).…”

Novel Insights into CB1 Cannabinoid Receptor Signaling: A Key Interaction Identified between the Extracellular-3 Loop and Transmembrane Helix 2

“…C-terminal truncation experiments from the Mackie laboratory (Jin et al, 1999) have shown that CB 1 (with truncation at C417) signals normally in the presence of agonists. With the exception of helix 8, the C terminus is largely unstructured, though recent work on an isolated C-terminal peptide suggests the existence of an additional C-terminal helix, helix 9 (Ahn et al, 2009b). However, recent results from the Mackie laboratory (Straiker et al, 2012) reinforce that the functional significance of the C terminus pertains to desensitization and receptor internalizationnot necessarily to receptor signaling by heterotrimeric G proteins.…”