In anterior pituitary cells or when transfected into host cell lines, the D2 dopamine receptor inhibits adenylyl cyclase and activates potassium channels. The GH-3 pituitary tumor cell line, which lacks functional D2 receptors, responds to epidermal growth factor (EGF) by expressing a D2 receptor that, paradoxically, couples to potassium channel activation but poorly inhibits adenylyl cyclase; this was correlated with a pronounced increase in alpha subunit of the G protein Gi3. In this study we have investigated the effects of EGF on the transduction mechanisms of D2 receptors in GH4C1 cells transfected and permanently overexpressing the rat short D2 receptor. Activation of D2 receptors in these cells resulted in both inhibition of adenylyl cyclase and opening of potassium channels and inhibition of prolactin release by both cyclic AMP-dependent and independent mechanisms. Exposure of the transfected GH4C1 cells to EGF caused a dramatic decrease in the coupling efficiency of the D2 receptor to inhibit cyclic AMP-dependent responses, leaving its activity toward potassium channels unchanged. The EGF treatment led to the concomitant increase in the membrane content of Gi3 protein. These results suggest that the transmembrane signaling specificity of G protein-coupled receptors can be modulated by the relative amounts of different G proteins at the cell membrane.