Salivary secretion-role of calcium

描述(申请人提供)：本申请的总体目标是阐明G蛋白偶联受体(GPCRs)和MAPK(MAPK)途径之间的串扰机制。需要检验的假设是：1)GPCRs通过MAPK途径传递信号，而不是通过参与EGF途径的分子/激酶，而是聚集在Ras/Raf-1/MEK上；2)MAPK途径反过来调节钙、cAMP和cPLA2信号，为调节细胞内信使水平提供反馈机制。具体目标是：鉴定和确定参与GPCR介导的MAPK激活的分子/激酶的作用：通过用特定抗体的Western blotting鉴定细胞裂解产物中的分子/激酶。M3受体介导的MAPK激活将通过确定钙、PKC、非受体酪氨酸激酶(TYKs)、P13-K、A-Kinase、G-Kinase和表皮生长因子受体(EGFR)的参与以及这些分子之间相互作用的生化证据来评估。激酶的激活将通过磷酸化和体外检测来实现。通过确定Rap1与B-Raf和MAPK通路成员的磷酸化和结合、与与Rap1相关的其他分子的相互作用以及EGFR的反调来评估由β-肾上腺素能受体(Beta-AR)介导的MAPK的激活。确定MAPK通路在GFCR诱导的信号转导中的作用：将TYKs与GPCR诱导的钙信号和淀粉酶释放联系起来的机制(S)将通过将M3诱导的钙释放/进入的变化与GQ/L 1、PLCGamma和1P3受体的酪氨酸磷酸化以及储存物清空相关联来评估。β-AR诱导的cAMP信号将通过将MAPK的cAMP调节与PDE4同工酶的刺激相关联来评估，PDE4同工酶将通过免疫沉淀和免疫印迹分析与特定的PDE4D抗体、PDE分析和淀粉酶释放来鉴定。确定MAPK通路在GPCR诱导的cPLA2激活和信号传递中的作用：cPLA2的MAPK激活将通过测量cPLA2的磷酸化来确定。AA诱导MAPK激活的机制将通过确定钙、PKC、TYKs和EGFR的参与来评估。TYKs在PLA2诱导的钙信号转导中的作用将通过将AA/代谢物诱导的钙释放/进入与PYK2、Src激酶和P13-K的酪氨酸磷酸化相关联来评估。通过将MAPK的激活与cAMP的合成和降解联系起来，来评估AA/代谢物介导的cAMP信号转导的机制/途径。

DESCRIPTION (provided by applicant): The overall goal of this application is to elucidate the mechanisms involved in the cross talk between G-protein-coupled receptors (GPCRs) and the MAP kinase (MAPK) pathway. The hypotheses to be tested are that 1) GPCRs signal through the MAPK pathway via molecules/kinases different from those involved in the EGF pathway, but converging on Ras/Raf-1/MEK and 2) the MAPK pathway, in turn, modulates calcium, cAMP, and cPLA2 signaling, providing a feedback mechanism for regulating intracellular messenger levels. Specific Aims are:Identification and determination of the role of molecules/kinases involved in GPCR-mediated activation of MAPK: Molecules/kinases will be identified in cell lysates by Western blotting with specific antibodies. M3 receptor-mediated activation of MAPK will be assessed by determining the involvement of calcium, PKC, non-receptor tyrosine kinases (TYKs), P13-K, A-Kinase, G-kinase, and the epidermal growth factor receptor (EGFR), and by biochemical evidence of an interaction between these molecules. Kinase activation will be by phosphorylation and in-vitro assays. Beta-adrenergic receptor (Beta-AR)-mediated activation of MAPK will be assessed by determining: phosphorylation and association of Rap 1 with B-Raf and members of the MAPK pathway, an interaction with other molecules associated with Rap 1, and by transmodulation of EGFR. Determination of the role of the MAPK pathway in GFCR-induced Signaling: The mechanism(s) linking TYKs to GPCR-induced calcium signaling, and amylase release will be assessed by correlating M3-induced changes in calcium release/entry with tyrosine phosphorylation of Gq/l 1, PLCgamma, and 1P3 receptors, and store emptying. Beta-AR-induced cAMP signaling will be assessed by correlating cAMP-regulation of MAPK with stimulation of PDE4 isoenzymes which will be identified by immunoprecipitation and immunoblot analysis with specific PDE4D antibodies, and PDE assays, and amylase release. Determination of the role of the MAPK pathway in GPCR-induced cPLA2 activation and signaling: MAPK activation of cPLA2 will be determined by measuring phosphorylation of cPLA2. Mechanisms involved in AA-induced MAPK activation will be assessed by determining the involvement of calcium, PKC, TYKs and EGFR. A role for TYKs in PLA2-induced calcium signaling will be assessed by correlating AA/metabolite-induced calcium release/entry with tyrosine phosphorylation of Pyk2, Src kinases and P13-K. Mechanisms/pathways involved in AA/metabolite-mediated cAMP signaling will be assessed by correlating MAPK activation with cAMP synthesis and degradation.