A chimeric D2 dopamine/m1 muscarinic receptor with D2 binding specificity mobilizes intracellular calcium in response to dopamine.
Using PCR methodology, a chimeric receptor cDNA was constructed in which the entire third cytoplasmic loop of the human D2 dopamine receptor was replaced by the analogous portion of the human m1 muscarinic receptor. When expressed in CHO cells, the chimeric D2/m1 receptor bound dopaminergic ligands with affinities similar to the native D2(414) receptor. Intracellular calcium levels (measured with Fura-2) were not altered when CHO cells expressing the D2(414) receptor were exposed to dopamine. In contrast, dopamine elevated intracellular calcium levels in cells expressing the D2/m1 chimeric receptor in a dose-dependent manner which was blocked by the D2 antagonist, fluphenazine. The ability to construct G-protein-linked receptor chimeras which mobilize calcium with nearly unaltered pharmacologic specificity raises the possibility of a generic strategy for creating non-radioisotopic reporter systems for use in drug discovery.
England
B P
BP
Affymax Research Institute, Palo Alto, CA 94304.
Ackerman
M S
MS
Barrett
R W
RW
D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D002678:Chimera; D006224:Cricetinae; D003412:Cricetulus; D004298:Dopamine / Q000494:pharmacology*; D016257:Fura-2; D006801:Humans; D008969:Molecular Sequence Data; D011869:Radioligand Assay; D011954:Receptors, Dopamine / Q000235:genetics / Q000378:metabolism*; D011976:Receptors, Muscarinic / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D014162:Transfection
D011954:Receptors, Dopamine; D011976:Receptors, Muscarinic; D002118:Calcium; D016257:Fura-2; D004298:Dopamine