A mutation in the second transmembrane region of the CB1 receptor selectively disrupts G protein signaling and prevents receptor internalization

We mutated a conserved aspartate in the second transmem-brane domain of the cannabinoid CB1 receptor to asparagine (D164N), stably transfected it into AtT20 cells, and examined the coupling of this mutant receptor to several intracellular effectors that are targets of wild-type CB1 receptor activation. We found that the D164N receptor binds the CB1 agonist WIN 55,212-2 with an affinity matching that of the wild-type CB1 receptor and inhibits Ca21 currents and cAMP production with an equivalent potency and efficacy. This mutation, however, blocks coupling of the receptor to the potentiation of inwardly rectifying potassium channel (KIR) currents and prevents inter-nalization of the receptor after exposure to agonist. Although the mutant receptor did not internalize, we found it was still capable of activating p42/44 MAP kinase. In addition, we mad