||Clinical treatment of severe pain primarily relies on the administration of opiate analgesics. Unfortunately these potent analgesics have a high propensity for developing tolerance and physical dependence. The newly discovered opioid receptor-like (ORL1) receptor has opened an exciting opportunity for the search of novel analgesics. Nociceptin, the endogenous ligand of ORL1 receptor is endowed with supraspinal pronociceptive/anti-opioid activity and affects pain perception. Antagonists of the ORL1 receptor are therefore predicted to suppress pain perception and may act as novel analgesics with low propensity for tolerance. To facilitate the screening of ORL1 receptor antagonist with inverse agonistic effect, a novel approach is being used. Specific point mutations introduced into G protein-coupled receptors (GPCR) are known to lead to constitutive activation of the receptor. Eleven potential sites for creating constitutively active ORL1 receptors were identified. One of the potential sites identified was able to turn ORL1 receptor into constitutively active state. Asn133 located in the third transmembrane was mutated to Trytophan. This N133W mutant receptor showed a significant increase over the wild type receptor in basal signaling through coupling to G16 and G14. This increase in basal signaling was shown to be dependent on the receptor density. A stable cell line expressing the N133W mutant (293/N133W) or the ORL1 receptors (293/ORL1) was constructed in HEK293 cells. When stimulated by forskolin, the cAMP accumulation response of 293/N133W cells was about 67 % of that observed in 293/ORL1 cells and the constitutive suppression of cAMP formation was found to be PTX-insensitive. The N133W receptor did not show enhanced potency for different classes of drugs in the PLC and cAMP assay. To further characterize the effect of this mutation on receptor signaling, extracellular signal regulated kinases (ERK1/2) activation was investigated. Result showed that the N133W mutant lost its ability to stimulate the phosphorylation of ERK1/2.This might be due to a negative feedback mechanism operating in the cell and chronic OFQ treatment was found to increase the basal activity of ERK1/2. Collectively, the present results indicated that (1) Asn133 to Trp mutation could create CAM ORL1 receptor. (2) There was no increase in potency of OFQ and Phe-OFQ on N133W mutant receptor. (3) The N133W mutant receptor lost its ability to stimulate the phosphorylation of ERK1/2. (4) Chronic OFQ treatment affects the basal ERK activity in both 293/ORL1 and 293/N133W cells.