Analysis of dopamine D2 receptor mutants deficient in arrestin binding using bioluminescence resonance energy transfer

Arrestins mediate dopamine D2 receptor (D2R) internalization, desensitization, and regulation of signaling proteins such as Akt and GSK3β. Lysine 149 (K149) and amino acids 212–215 in intracellular loops two and three of D2R, respectively, are important for the interaction between arrestin3 and D2R. Our objective is to determine the smallest mutation that prevents arrestin binding while retaining other D2R functions. The interaction between D2R and arrestin3 was evaluated using bioluminescence resonance energy transfer (BRET). The D2R agonist quinpirole enhanced the BRET signal in cells expressing wild type D2R or D2R‐K149C. Mutation of residues 212–215 abolished arrestin recruitment, but resulted in low membrane expression of the receptor. Mutation of amino acids 214 and 215 significantly decreased arrestin recruitment while retaining normal membrane expression. In conclusion, mutations in D2R intracellular loop three reduce arrestin binding. In contrast to our previous result using a different assay, arrestin binding was maintained in cells expressing D2R‐K149C, perhaps reflecting a mutation‐induced alteration in the time course of arrestin recruitment. (VHA, MH045372 , and DA007262 )