Abstract

We mutated a conserved aspartate in the second transmembrane 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 Ca²⁺ 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 internalization 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 made a reciprocal mutation that exchanged the aspartate with an asparagine in the seventh transmembrane region (D164N/N394D). In other seven-membrane-spanning receptors, this reciprocal mutation is known to restore functions disrupted by the mutation of the single conserved aspartate. However, activation of D164N/N394D did not potentiate KIR current, nor did it internalize. We conclude that D164 is necessary for potentiation of KIR current and internalization of receptor but not necessary for agonist binding, inhibition of cAMP production, inhibition of Ca²⁺ currents, or activation of p42/44 MAP kinase. Furthermore, CB₁receptor internalization is not necessary for MAP kinase activation.