Dopamine D2 receptor isoforms expressed in AtT20 cells differentially couple to G proteins to acutely inhibit high voltage-activated calcium channels

The dopamine D2 receptor belongs to the serpentine superfamily of receptors, which have seven transmembrane segments and activate G proteins. D2 receptors are known to be linked, through Galpha(o)- and Galpha(i)-containing G proteins, to several signaling pathways in neuronal and secretory cells, including inhibition of adenylyl cyclase and high voltage-activated Ca2+ channels (HVA-CCs). The dopamine D2 receptor exists in two alternatively spliced isoforms, "long" and "short" (D2L, and D2S, respectively), which have identical ligand binding sites but differ by 29 amino acids in the third intracellular loop, the proposed site for G protein interaction. This has led to the speculation that the two isoforms may interact with different G proteins. We have transfected the AtT20 cell line with either D2L (KCL line) or D2S (KCS line) to facilitate experimentation on the individual isoforms. Both lines show dopamine agonist-dependent inhibition of Q-type HVA-CCs. We combined G protein antisense knock-down studies with multiwavelength fluorescence video microscopy to measure changes in HVA-CC inhibition to investigate the possibility of differential G protein coupling to this inhibition. The initial, rapid, K+ depolarization-induced increase in intracellular Ca2+ concentration is due to influx through HVA-CCs. Our studies reveal that both D2 isoforms couple to Galpha(o) to partially inhibit this influx. However, D2L also couples to Galpha(i)3, whereas D2S couples to Galpha(i)2. These data support the hypothesis of differential coupling of D2 receptor isoforms to G proteins.