CAMP DEPENDENT PROTEIN KINASES AS MEDIATORS OF RECEPTOR ACTION

不同神经递质的不同生化效应 刺激cAMP产生的荷尔蒙被认为是 通过对特定的PKA亚型进行特定部位的本地化。 II型PKA的定位是通过蛋白质-蛋白质来维持的 调控亚单位(RII)与特异性A-激酶的相互作用 锚定蛋白(AKAP)。在过去的资助期内，我们有 演示了PKA锚定是通过 AKAP上带有两亲性螺旋的RII二聚体，阐明了它们的作用 将PKA靶向特定亚细胞的所选AKAP的数量 并证明了PKA锚定的破坏 解偶联细胞内的某些cAMP反应事件。未来的计划 下一个资助期的重点是扩大这些调查范围。目标1 将通过测量来从结构上阐明RII上的AKAP结合部位 AKAP与AKAP结合亲和力的变化 在选定的RII突变体中的平衡透析和通过完成 RII片段与AKAP多肽络合时的结构 多维核磁共振。目标2将定义过氧化物体靶向 AKAP220上的序列，一种新的AKAP，与 过氧酶体。目标3将确定是否操纵PKA本地化 通过确定正确的PKA是否正确来改变CAMP反应事件 锚定有助于两个生理靶点的磷酸化： 参与调控的转录因子CREB 基因表达，以及MAP激酶级联反应的酶 调节细胞生长。

The diverse biochemical effects of different neurotransmitters or hormones that stimulate cAMP production have been proposed to occur through the site-specific localization of particular PKA subtypes. Localization of the type II PKA is maintained through protein-protein interactions between the regulatory subunit (RII) and specific A-kinase anchoring proteins (AKAPs). During the past funding period we have demonstrated that PKA anchoring proceeds through interaction of the RII dimer with an amphipathic helix on the AKAP, elucidated the roles of selected AKAPs in targeting PKA to specific subcellular compartments, and demonstrated that disruption of PKA anchoring uncouples certain cAMP-responsive events inside cells. Plans for the next funding period focus on extending these lines of inquiry. Aim 1 will structurally elucidate the AKAP-binding site on RII by measuring changes in AKAP-binding affinity by plasmon resonance and equilibrium dialysis in selected RII mutants and by completing the structure of the RII fragment when complexed to an AKAP peptide by multidimensional NMR. Aim 2 will define the peroxisomal targeting sequence on AKAP220, a novel AKAP which associates with peroxisomes. Aim 3 will establish if manipulation of PKA localization alters cAMP-responsive events by determining whether correct PKA anchoring facilitates the phosphorylation of two physiological targets: the transcription factor CREB which is involved in the regulation of gene expression, and enzymes of the MAP kinase cascade which modulate cell growth.