Anavi-Goffer Sharon, Fleischer Daniel, Hurst Dow P, Lynch Diane L, Barnett-Norris Judy, Shi Shanping, Lewis Deborah L, Mukhopadhyay Somnath, Howlett Allyn C, Reggio Patricia H, Abood Mary E
California Pacific Medical Center Research Institute, San Francisco, California 94107, USA.
J Biol Chem. 2007 Aug 24;282(34):25100-13. doi: 10.1074/jbc.M703388200. Epub 2007 Jun 26.
The intracellular C-terminal helix 8 (H8) of the CB(1) cannabinoid receptor deviates from the highly conserved NPXXY(X)(5,6)F G-protein-coupled receptor motif, possessing a Leu instead of a Phe. We compared the signal transduction capabilities of CB(1) with those of an L7.60F mutation and an L7.60I mutation that mimics the CB(2) sequence. The two mutant receptors differed from wild type (WT) in their ability to regulate G-proteins in the [(35)S]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 Ca(2+) 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 Galpha(i3) but not Galpha(0A) in the L7.60I mutant, whereas the reduction in the time course for the L7.60F mutant was governed by Galpha(i3). Furthermore, Galpha(i3) but not Galpha(0A) enhanced basal facilitation ratio, suggesting that Galpha(i3) is responsible for CB(1) tonic activity. Co-immunoprecipitation studies revealed that both mutant receptors were associated with Galpha(i1) or Galpha(i2) but not with Galpha(i3). Molecular dynamics simulations of WT CB(1) 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)(5,6)L contributes to maximal activity of the CB(1) receptor and provides a molecular basis for the differential coupling observed with chemically different agonists.
CB(1)大麻素受体的细胞内C末端螺旋8（H8）偏离了高度保守的NPXXY(X)(5,6)F G蛋白偶联受体基序，其具有亮氨酸而非苯丙氨酸。我们将CB(1)与模拟CB(2)序列的L7.60F突变体和L7.60I突变体的信号转导能力进行了比较。在[³⁵S]鸟苷5'-3-O-(硫代)三磷酸结合试验中，这两种突变受体在调节G蛋白的能力上与野生型（WT）不同。L7.60F受体对激动剂WIN-55,212-2和CP-55,940的刺激减弱，但对HU-210无此现象，而L7.60I受体对所有测试激动剂以及反向激动剂利莫那班的刺激均受损。突变体比WT受体内化更快，但在从生长抑素受体隔离G蛋白方面能力相当。WIN-55,212-2对N型钙电流的抑制时间进程和最大抑制程度在突变体中均降低。用百日咳毒素不敏感的G蛋白进行的重组实验表明，L7.60I突变体中与Gαi3而非Gα0A的偶联丧失，而L7.60F突变体时间进程的缩短由Gαi3控制。此外，Gαi3而非Gα0A增强了基础易化率，表明Gαi3负责CB(1)的张力活性。免疫共沉淀研究表明，两种突变受体均与Gαi1或Gαi2相关，而与Gαi3无关。在1-棕榈酰-2-油酰磷脂酰胆碱双层膜中对WT CB(1)受体和每个突变体进行的分子动力学模拟表明，每个H8的堆积情况不同。每个H8沿螺旋主链的氢键模式也不同，H8（R7.56(400)-K7.58(402)）肘部区域的几何形状也不同。本研究表明，对NPXXY(X)(5,6)L的进化修饰有助于CB(1)受体的最大活性，并为用化学性质不同的激动剂观察到的差异偶联提供了分子基础。
