RUI: Regulation of MAP Kinase and NHE1 Activation by the G Proteins Gq and G13

Minnesota State University Moorhead, Moorhead MN

Investigators

Abstract

The Na + -H + exchanger isoform 1 (NHE1) is present in virtually all mammalian cells where it is involved in the regulation of intracellular pH (pHi) and cellular volume. NHE1 activity can be modulated by a wide variety of intracellular signaling pathways. The receptors that activate the pathways and the specific protein kinases involved in the regulation vary dramatically in different cell types. To date there has been no complete dissection of the a 1 adrenergic signaling pathway in Chinese hamster lung (CCL39) fibroblasts. The overall goal of this project is to clarify the signaling pathway for the regulation of NHE1 in CCL39 cells. Also to clarify which intermediates are involved in the signaling pathway. The following aims are proposed: Specific Aim 1: To study the activation of MAPK and NHE1 by PE. Preliminary studies indicate that phenylephrine (PE) can activate both MAPK and NHE1. The time course and the dose response MAPK activation will be investigated.The relationship between the activation of MAPK and the stimulation of NHE1 will also be investigated. Specific activators and inhibitors of the MAPK pathway will be used to accomplish this task. This aim will clarify the role of MAPK as a potential key intermediate in the stimulation of NHE by PE. Specific Aim 2: To study the involvement of PKC and Ras in the PE induced activation of MAPK and NHE1. Preliminary studies indicate that PE stimulates both MAPK and PLD. Published data indicates that a 1-adrenergic receptors activate Gq. This activation can lead to a q stimulating PLCbeta and leads to the activation of PKC. Additionally, the Gq . subunit can lead to the activation of Ras. To discern the regulatory pathways by which PE modulates NHE1 activity, the time course and dose response for activation of PKC and Ras will be determined. It will then be determined if MAPK and NHE1 activation dependent on one or both of the intermediates. Transfected cells expressing the dominant/negative Ras and/or dominant/negative Raf-1 will be used to determine if activation of MAPK and NHE1 by PE functions occurs through a Ras-dependent or independent mechanism. Expression of these proteins will disrupt the postulated signaling pathways at different levels clarifying the points of convergence of the pathways. This aim will determine the signaling molecules involved in the PE activation of MAPK and NHE1 and clarify the relationship between the signaling pathways activated by PE. Specific Aim 3: To expand the involvement of undergraduates in meaningful research. The primary purpose for faculty members of the biology and chemistry departments at Moorhead State University (MSU) is to have ongoing research projects that involve undergraduates in a meaningful research experience. Students have been trained to perform all the procedures required to carryout the investigations of the signaling pathways and to measure NHE activity as outlined in the proposal. This research project will allow 8 - 10 students to be involved in research during the academic year and three students to work full-time during the summer months of the three years covered by the grant.