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Characterization of the Physical and Functional Interaction between a Novel Protein CRIPla and the CBj Cannabinoid ReceptorCBi receptors modulate synaptic transmission and play important roles in analgesia, appetite and neuroprotection. However, little is known about how CBi activity is regulated. The possibility that a novel protein CRIPla interacts with CBj was studied to determine whether CBt functions are modulated by such an interaction. CRIPla specifically interacted with the C-terminus of CBi in a GST pull-down assay and co-precipitated with CBi oligomer in HEK293 cells, demonstrating that CRIPla interacts with CB^ Moreover, CRIPla and CBj co-localized when heterologously expressed in HEK293 cells and in rat superior cervical ganglion (SCG) neurons. The functional CBr CRIPla interaction was investigated using whole-cell voltage-clamp recordings of N-type Ca2+ channels in SCG neurons heterologously expressing CBi with or without CRIPla. The electrophysiological data demonstrated that CRIPla significantly reduced the ability of the CB! inverse agonist SR141716 to enhance the Ca2+ current but did not affect the ability of the CBi agonist WIN 55,212- 2 to inhibit the Ca2+ current. In addition, CRIPla significantly decreased Ca2+ current basal facilitation ratio. Since CRIPla did not alter CB! expression pattern or the EC50 response to WIN 55,212-2 in neurons co-expressing CRIPla and CBi; it is unlikely that the attenuated SR141716 response or the reduced Ca2+ current basal facilitation ratio resulted from decreased surface expression of CB,. Our data indicate that CRIPla inhibits the constitutive activity of CB! receptors. Deletion of the last nine amino acids of the CBi receptor abolished the interaction with CRIPla in a GST pull-down assay, indicating that these residues of the CBi receptor constitute the CRIPla interaction domain. In SCG neurons, the CBiA465-473 receptor missing the CRIPla interaction domain was constitutively active but the constitutive activity was not affected by CRIPla, demonstrating that deletion of the CRIPla interaction domain from CB! receptors reversed the ability of CRIPla to inhibit CBi constitutive activity. Taken together, our data suggest that CRIPla controls CBi constitutive activity by interacting with the last nine amino acids of the CBi receptor.