A site‐directed mutagenesis study on the conserved alanine residue in the distal third intracellular loops of cholecystokinin B and neurotensin receptors

An alanine residue at the C‐terminal tail of the third intracellular loop is highly conserved among various G q protein‐coupled receptors including rat cholecystokinin B (CCK B ) and neurotensin receptors. To investigate the functional significance of the conserved alanine in the activation of G q proteins and phospholipase C (PLC) by CCK B and neurotensin receptors, the alanine residue was mutated in the present study. Subsequently, the ability of resulting mutant receptors to activate PLC was investigated by measuring the formation of inositol phosphates (IP) in COS‐7 cells and recording Ca 2+ ‐activated chloride currents from Xenopus oocytes. Site‐directed mutagenesis was performed to mutate alanine at position 332 of rat CCK B receptor to glutamate. When the (A332E) mutant receptor was expressed in COS‐7 cells and Xenopus oocytes, the efficacy and the potency of sulphated cholecystokinin octapeptide (CCK‐8) to stimulate polyphosphoinositide hydrolysis in COS‐7 cells and evoke calcium‐dependent Cl − currents in oocytes were not significantly affected. Alanine residue at position 302 of rat neurotensin receptor was also mutated to glutamate. When expressed in COS‐7 cells and Xenopus oocytes, the resulting (A302E) mutant receptor was strongly defective in stimulating phosphatidylinositol turnover in COS‐7 cells and evoking Ca 2+ ‐dependent chloride currents in oocytes. In summary, the present study demonstrates that alanine residue at the C‐terminus of third cytoplasmic domain is required for the full activation of G q proteins and PLC by neurotensin receptors. However, in contrast to other G q protein‐coupled receptors, alanine at the distal third intracellular loop does not play a significant role in CCK B receptor activation of PLC.British Journal of Pharmacology (1997) 121 , 310–316; doi: 10.1038/sj.bjp.0701117