Judy Barnett-Norris

A critical role for a tyrosine residue in the cannabinoid receptors for ligand recognition

Previous mutation and modeling studies have identified an aromatic cluster in the transmembrane helix (TMH) 3-4-5 region as important for ligand binding at the CB(1) and CB(2) cannabinoid receptors. Through novel mixed mode Monte Carlo/Stochastic Dynamics (MC/SD) calculations, we tested the importance of aromaticity at position 5.39(275) in CB(1). MC/SD calculations were performed on wild-type (WT) CB(1) and two mutants, Y5.39(275)F and Y5.39(275)I. Results indicated that while the CB(1) Y5.39(275)F mutant is very similar to WT, the Y5.39(275)I mutant shows pronounced topology changes in the TMH 3-4-5 region. Site-directed mutagenesis studies of tyrosine 5.39 to phenylalanine (Y-->F) or isoleucine (Y-->I) in both CB(1) and CB(2) were performed to determine the functional role of this amino acid in each receptor subtype. HEK 293 cells transfected with mutant receptor cDNAs were evaluated in radioligand binding and cyclic AMP assays. The CB(1) mutant and WT receptors were also co-expressed with G-protein-coupled inwardly rectifying channels (GIRK1 and GIRK4) in Xenopus oocytes to assess functional coupling. The Y-->F mutation resulted in cannnabinoid receptors with subtle differences in WT binding and signal transduction. In contrast, the Y-->I mutations produced receptors that could not produce signal transduction or bind to multiple cannabinoid compounds. However, immunofluorescence data indicate that the Y-->I mutation was compartmentalized and expressed at a level similar to that of the WT cannabinoid receptor. These results underscore the importance of aromaticity at position CB(1) 5.39(275) and CB(2) 5.39(191) for ligand recognition in the cannabinoid receptors.

Helix 8 Leu in the CB1 cannabinoid receptor contributes to selective signal transduction mechanisms.

The intracellular C-terminal helix 8 (H8) of the CB1 cannabinoid receptor deviates from the highly conserved NPXXY(X)5,6F G-protein-coupled receptor motif, possessing a Leu instead of a Phe. We compared the signal transduction capabilities of CB1 with those of an L7.60F mutation and an L7.60I mutation that mimics the CB2 sequence. The two mutant receptors differed from wild type (WT) in their ability to regulate G-proteins in the [35S]guanosine 5′-3-O-(thio)triphosphate binding assay. The L7.60F receptor exhibited attenuated stimulation by agonists WIN-55,212-2 and CP-55,940 but not HU-210, whereas the L7.60I receptor exhibited impaired stimulation by all agonists tested as well as by the inverse agonist rimonabant. The mutants internalized more rapidly than WT receptors but could equally sequester G-proteins from the somatostatin receptor. Both the time course and maximal N-type Ca2+ current inhibition by WIN-55,212-2 were reduced in the mutants. Reconstitution experiments with pertussis toxin-insensitive G-proteins revealed loss of coupling to Gαi3 but not Gα0A in the L7.60I mutant, whereas the reduction in the time course for the L7.60F mutant was governed by Gαi3. Furthermore, Gαi3 but not Gα0A enhanced basal facilitation ratio, suggesting that Gαi3 is responsible for CB1 tonic activity. Co-immunoprecipitation studies revealed that both mutant receptors were associated with Gαi1 or Gαi2 but not with Gαi3. Molecular dynamics simulations of WT CB1 receptor and each mutant in a 1-palmitoyl-2-oleoylphosphatidylcholine bilayer suggested that the packing of H8 is different in each. The hydrogen bonding patterns along the helix backbones of each H8 also are different, as are the geometries of the elbow region of H8 (R7.56(400)-K7.58(402)). This study demonstrates that the evolutionary modification to NPXXY(X)5,6L contributes to maximal activity of the CB1 receptor and provides a molecular basis for the differential coupling observed with chemically different agonists.