A calcineurin homologous protein inhibits GTPase-stimulated Na-H exchange.

Abstract

Activation of the ubiquitously expressed Na-H exchanger, NHE1, results in an increased efflux of intracellular HW. The increase in intracellular pH associated with this H+ efflux may contribute to regulating cell proliferation, differentiation, and neoplastic transformation. Although NHE1 activity is stimulated by growth factors and hormones acting through multiple GTPase-mediated pathways, little is known about how the exchanger is directly regulated. Using expression library screening, we identified a novel protein that specifically binds to NHE1 at a site that is critical for growth factor stimulation of exchange activity. This protein is homologous to calcineurin B and calmodulin and is designated CHP for calcineurin B homologous protein. Like NHE1, CHP is widely expressed in human tissues. Transient overexpression of CHP inhibits serum-and GTPase-stimulated NHEl activity. CHP is a phosphoprotein and expression of constitutively activated GTPases decreases CHP phosphorylation. The phosphorylation state of CHP may therefore be an important signal controlling mitogenic regulation of NHE1.

Mutations of Arg440 and Gly455/Gly456 Oppositely Change pH Sensing of Na+/H+ Exchanger 1

“…We showed that CHP is tightly bound to a short juxtamembrane segment of the exchanger (amino acids 510 -530 of subdomain I in the case of NHE1) (6). Interestingly, in addition to the large reduction of V max , CHP binding-defective NHE1 mutants exhibited a marked shift of the pH i dependence to an acidic side.…”

“…Recently, we have reported that the calcineurin B homologous protein CHP is an essential co-factor for the expression of high physiological levels of exchange activity in many plasma membrane NHE isoforms (6). We showed that CHP is tightly bound to a short juxtamembrane segment of the exchanger (amino acids 510 -530 of subdomain I in the case of NHE1) (6).…”

“…Regulation of NHE1 by these stimuli has been reported to occur through a variety of signaling molecules, i.e. calcinuerin B homologous protein (6,7), Ca 2ϩ /calmodulin (8,9), low molecular mass GTPases Ras and Rho (10 -12), p42/44 mitogen-activated protein kinases (13), p90 ribosomal S6 kinase (14), 14-3-3 protein (15), Nck-interacting kinase (16), and phosphatidylinositol 4,5-bisphosphate (17). However, the interrelationship of these signaling molecules and the mechanism by which they modulate the interaction of the putative H ϩ modifier site with intracellular protons is still unclear.…”

Na+/H+ Exchangers of Mammalian Cells

“…CHP appears to be constitutively phosphorylated, and stimulation of transport is accompanied by its dephosphorylation. This prompted the suggestion that the phosphoprotein is normally associated with the antiporter, thereby exerting a suppressive effect, and that dissociation upon dephosphorylation may lead to activation of Na ϩ /H ϩ exchange (55). Independent experiments detected a polypeptide of Ϸ24 kDa, the approximate size of CHP, constitutively associated with NHE1 in several cell types (56).…”

“…1 and Ref. 55). Binding of CHP exerts an inhibitory effect on NHE1, although it remains unclear whether Ca 2ϩ is required for this interaction.…”