Received for publication August 23, 2006.
Revised September 30, 2006.
Accepted for publication October 17, 2006.

Ca²⁺-calmodulin and Jak2 are Required for Activation of Sodium-proton Exchange by the G_i-coupled 5-hydroxytryptamine_1A Receptor

Abstract

The type I sodium-proton exchanger (NHE-1) is expressed ubiquitously and regulates key cellular functions including mitogenesis, cell volume, and intracellular pH. Despite its importance, the signaling pathways that regulate NHE-1 remain incompletely defined. In this work, we present evidence that stimulation of the 5-hydroxytryptamine_1A (5-HT_1A) receptor results in the formation of a signaling complex that includes activated Jak2, Ca²⁺/calmodulin (CaM), and NHE-1, and which involves tyrosine phosphorylation of CaM. The signaling pathway also involves rapid agonist-induced association of CaM and NHE-1 as assessed by co-immunoprecipitation studies, and by bioluminescence resonance energy transfer studies in living cells. We propose that NHE-1 is activated through this pathway: 5-HT_1A receptor --> G_i2 and/or G_i3 --> Jak2 activation --> tyrosine phosphorylation of CaM --> increased binding of CaM to NHE-1 --> induction of a conformational change in NHE-1 that unmasks an obscured proton-sensing and/or proton transporting region of NHE-1 --> activation of NHE-1. The G_i/o-coupled 5-HT_1A receptor now joins a handful of G_q-coupled receptors and hypertonic shock as upstream activators of this emerging pathway. In the course of this work, we have presented clear evidence that CaM can be activated through tyrosine phosphorylation in the absence of a significant role for elevated intracellular Ca²⁺. We have also demonstrated for the first time that the association of CaM with NHE-1 in living cells is a dynamic process.

Key words: Janus kinase, calcium, calmodulin, proton, serotonin receptor, signal transduction