Desensitization of the human D1 dopamine receptor: Evidence for Involvement of both cyclic AMP-dependent and receptor-specific protein kinases

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Abstract

Human SK-N-MC neurotumor cells express D1 dopamine receptors coupled to adenylyl cyclase. Following exposure of the cells to dopamine, there was a time- and dose-dependent loss of dopamine responsiveness that involved both a reduction in the maximum response (Vmax) and a shift to less sensitivity in the dose response (Kact). Although the shift in Kact occurred more rapidly than the reduction in Vmax, both effects were completed within a 20-min exposure of the cells to dopamine. During this rapid period of desensitization, there was no loss of D1 receptors from membranes prepared from dopamine-treated cells, but there was a reduction in the proportion of agonist high-affinity binding sites. More prolonged exposure to D1 agonists led to a progressive loss of binding activity. The desensitization was homologous as exposure of the cells, which have β1-adrenergic receptors, to isoproterenol did not alter their response to dopamine. A shift in Kact but not in Vmax was observed when membranes from control cells were incubated with dopamine, ATP, and the catalytic subunit of cyclic AMP-dependent protein kinase (PKA). To pursue the role of protein kinases in the desensitization process, cells were transiently made permeable, loaded with a PKA inhibitor or with heparin, an inhibitor of the β-adrenergic receptor kinase, and exposed to dopamine. The PKA inhibitor blocked the shift in Kact, whereas heparin inhibited the reduction in Vmax. Our results suggest that desensitization of human D1 dopamine receptors involves both PKA and a receptor-specific kinase and that the action of both kinases in intact cells requires agonist-occupied receptors.

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