Synapses are central players in neuronal signal transmission and prime targets for drug treatments of various neurological and mental disorders. The long-term goal of this application is to characterize the assembly and function of signaling pathways at postsynaptic sites. This proposal focuses on molecular mechanisms that target PKA together with its regulator (beta2 adrenergic receptor, beta2 AR) and its antagonist (phosphatase PP2A) to postsynaptic effector proteins, in particular the L-type Ca2+ channel Cav1.2. PKA upregulates Cav1.2 currents. Cav1.2 is clustered at postsynaptic sites. We discovered that Cav1.2 assembles the beta2 AR, the trimeric Gs protein, adenylyl cyclase, AKAP75/150, PKA and the counter- balancing phosphatase PP2A into a macromolecular signaling complex. Our single channel recordings revealed that upregulation of channel activity upon stimulation of beta2 AR is highly localized in neurons (Science 293, 98-101). This is the very first example of a complex that links a cAMP-coupled receptor to one of its ultimate targets. This localized signaling explains how the diffusible cAMP can mediate specific effects. The interaction sites of the channel with p2 AR, AKAP75/150, and PP2A, will be defined and mutated in a nifedipine-insensitive T1039Y Cav1.2 mutant and expressed in primary hippocampal cultures as will be S1928 (to alanine), the main PKA site. Localized regulation of these mutants by the beta2 AR - PKA pathway will be measured by cell-attached single channel recordings and imaging of Ca2+ transients in dendritic spines with nifedipine present to inhibit endogenous L-type channels. Complementary studies with AKAP150 KO mice and with (poly)peptides that disrupt the beta2 AR interactions with endogenous Cav1.2 will be performed. The physiological relevance of PP2A binding with the C-terminus of Cav1.2 will be analyzed in an analogous way. An increase in Cav1.2 channel activity as mediated by PKA has been implicated in depression and anxiety disorders and contributes to the etiology of Alzheimer's disease. The postsynaptic assembly of specific signaling components that control PKA-mediated phosphorylation of Cav1.2 constitutes, therefore, a potentially effective and specific target for drugs that disrupt some of these interactions, thereby inhibiting adverse signaling events under pathological conditions.
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