Role of protein phosphorylation and dephosphorylation in activation and desensitization of the cAMP-dependent Na+/H+ antiport.

Abstract

The Na+/H+ antiporter of trout erythrocytes is activated by agents raising intracellular cAMP, whereas other Na+/H+ exchangers are insensitive to or inhibited by cAMP. Cloning of the beta agonist-activated exchanger (beta NHE) reveals the presence of two consensus sites for phosphorylation by the cAMP-dependent protein kinase A (cAMP-PKA) on the cytoplasmic loop. Transfected to fibroblasts, beta NHE can no longer be activated by cAMP when these consensus sites are removed, indicating regulation through cAMP-PKA. Moreover, it has been shown that activation of the exchanger is rapidly followed by its desensitization. To further investigate the role of phosphorylation in these processes, we examined the effects of protein kinase and phosphatase inhibitors on the antiporter activation and desensitization in trout red cells. Na+/H+ exchange was not induced by strong acidification, indicating that beta NHE is normally in a nonfunctional state, whereas cAMP did activate the system by forcing beta NHE into a functional conformation; preincubation of cells with the kinase inhibitor H89 blocked cAMP-activation, confirming the role of cAMP-PKA in the activation process. The protein phosphatase inhibitor okadaic acid (OA) neither activated the exchange when added on unstimulated cells nor prevented deactivation of beta agonist-activated beta NHE by propranolol. Hence, the cAMP-dependent phosphorylation involved in the activating process is controlled by an OA-insensitive phosphatase. beta NHE activated by beta agonist or cAMP shifts rapidly into a refractory state, accounting for the previously described desensitization. Desensitization was blocked and reversed by OA, indicating a control by an OA-sensitive phosphatase of the phosphorylation level of a site critical for the desensitizing process. Phosphorylation of this (site 2) and of the activating site (site 1) is mediated by cAMP-PKA, as demonstrated by the effects of both intracellular cAMP concentration and kinase inhibitor H89 on the Na+/H+ exchange activity. Based on these data, we proposed that beta NHE can exist in three different states (inactive I, activated A, and desensitized D). Conversion of I to A needs the simultaneous phosphorylation by cAMP-PKA of sites 1 and 2. These two sites might constitute the two neighboring cAMP-PKA sites located on the cytoplasmic loop as deduced from the oligonucleotide sequence. Dephosphorylation of site 2 and subsequent binding of an arrestin-like protein are assumed to account for desensitization of the antiport.(ABSTRACT TRUNCATED AT 400 WORDS)