Biochemical and pharmacological studies on dopamine D1 and D2L receptors

grantor: University of TorontoD1 and D2L receptors expressed in Sf9 cells were pharmacologically indistinguishable from native receptors, and were shown for the first time to be posttranslationally modified by phosphorylation and palmitoylation. Further, direct evidence now showed the existence of dopamine receptor dimers, in equilibrium with receptor monomers, formed by intermolecular noncovalent interactions involving TM regions. The exposure of D1/cells to dopamine for 15 min resulted in the desensitization of the D1 receptor stimulated adenylyl cyclase system, which was attributed to the increase in phosphorylation and palmitoylation level of the receptor and uncoupling of the receptor from G protein. Following 60 min of agonist exposure, surface D1 receptors, comprising predominantly receptor monomers, were internalized into an intracellular light vesicular membrane fraction. Pretreatment of D1/cells with concanavalin A or sucrose completely blocked agonist-induced D1 receptor internalization without preventing agonist-induced desensitization indicating that these processes were temporally and biochemically distinct mechanisms regulating D1 receptor function. In marked contrast, the D2L receptor coupled adenylyl cyclase system was resistant to agonist-induced desensitization, and dopamine exposure mediated a paradoxical increase in surface D2L receptor density, involving translocation of intracellular receptors and possibly receptor dimerization. In cells coexpressing D1 and D2L receptors, no modulation of D1 binding in the presence of the D2L receptor, and no modulation of D2L binding in the presence of the D1 receptor, or D1 and D2L receptor heterodimerization was observed. However, in tissues containing both D1 and D2L receptors, selective activation of these receptors resulted in a synergistic adenylyl cyclase response. Under similar conditions, when dopamine was the activator, effector activity was the net effect of D1 receptor stimulation and D2L receptor inhibition. In summary, these findings showed for the first time that D1 and D2L receptors were posttranslationally modified, exist as dimers, and are subject to distinct patterns of regulation upon exposure to dopamine. These processes have not been demonstrated previously, and should prove to be of significant fundamental importance for understanding the function of D1 and D2L and possibly other G protein-coupled receptors.Ph.D