The overall objective of this proposal is to determine signaling networks mediating the inhibitory actions of the somatostatin receptor subtype SSTR1. SSTR1 has been found to regulate previously undescribed targets: the ubiquitously expressed Na-H exchanger, NHE1, a novel NHE1- regulatory protein, CHP, the GTPase G alpha 12, and the cytoskeletal proteins vinculin, tensin, and actin. During the previous funding period, somatostatin inhibition of NHE1 was found to be mediated by SSTR1, 3 and 4, but not by SSTR2 or 5, and chimeric SSTR were used to identify amino acids that might confer this inhibition. GTPases coupled to the stimulation and inhibition of NHE1 were identified, and a novel NHE1-inhibitory protein, CHP (calcineurin homologous protein), was sequenced. SSTR1 regulates the phosphorylation of CHP, suggesting that CHP acts in a signaling pathway downstream of this receptor. CHP and NHE1 regulate cell proliferation and their regulation by SSTR1 may be a mechanism mediating SSTR1 inhibition of cell growth. During the previous funding period, SSTR1 was also found to inhibit the formation of focal complexes, cell adhesion, and the phosphorylation and polymerization of actin. Changes in the actin cytoskeleton can regulate secretory events and cell growth. The current proposal will investigate how SSTR1 regulates these newly identified targets and how these targets might contribute to SSTR1 inhibition of specific cell functions. The unifying hypothesis of this proposal is that multiple signaling mechanisms cooperate to mediate the inhibitory actions of SSTR1. This hypothesis will be investigated through two specific aims.
In Aim 1, SSTR1 inhibition of NHE1 activity will be investigated by determining whether inhibition of NHE1 is mediated by receptor subtype-specific amino acid motifs, G alpha 12, and the novel NHE1-regulatory protein CHP, and by determining whether NHE1 and CHP mediate SSTR1 inhibition of cell growth.
In Aim 2, SSTR1 regulation of the actin cytoskeleton will be investigated by determining whether focal adhesion kinases, GTPases and CHP mediate SSTR1 inhibition of cell adhesion and actin phosphorylation. Additionally, the functional importance of SSTR1- regulated cytoskeletal effects will be studied by determining whether this action is shared by SSTR2-5 and whether cytoskeletal regulatory proteins modulate hormone secretion. Identifying a cooperative action between the actin cytoskeleton, CHP, NHE1, and cell growth and secretion has significant implications that extend beyond the immediate focus of SSTR1 by contributing to our understanding of how these signals control cell functions.
