ROLE OF PROTEIN-PHOSPHORYLATION AND DEPHOSPHORYLATION IN ACTIVATION AND DESENSITIZATION OF THE CAMP-DEPENDENT NA+ H+ ANTIPORT/

The Na+/H+ antiporter of trout erythrocytes is activated by agents rai sing intracellular cAMP, whereas other Na+/H+ exchangers are insensiti ve to or inhibited by cAMP. Cloning of the beta agonist-activated exch anger (betaNHE) reveals the presence of two consensus sites for phosph orylation by the cAMP-dependent protein kinase A (cAMP-PKA) on the cyt oplasmic loop. Transfected to fibroblasts, betaNHE can no longer be ac tivated by cAMP when these consensus sites are removed, indicating reg ulation through cAMP-PKA. Moreover, it has been shown that activation of the exchanger is rapidly followed by its desensitization. To furthe r investigate the role of phosphorylation in these processes, we exami ned the effects of protein kinase and phosphatase inhibitors on the an tiporter activation and desensitization in trout red cells. Na+/H+ exc hange was not induced by strong acidification, indicating that betaNHE is normally in a nonfunctional state, whereas cAMP did activate the s ystem by forcing betaNHE into a functional conformation; preincubation of cells with the kinase inhibitor H89 blocked cAMP-activation, confi rming the role of cAMP-PKA in the activation process. The protein phos phatase inhibitor okadaic acid (OA) neither activated the exchange whe n added on unstimulated cells nor prevented deactivation of beta agoni st-activated betaNHE by propranolol. Hence, the cAMP-dependent phospho rylation involved in the activating process is controlled by an OA-ins ensitive phosphatase. BetaNHE activated by beta agonist or cAMP shifts rapidly into a refractory state, accounting for the previously descri bed desensitization. Desensitization was blocked and reversed by OA, i ndicating a control by an OA-sensitive phosphatase of the phosphorylat ion level of a site critical for the desensitizing process. Phosphoryl ation of this (site 2) and of the activating site (site 1) is mediated by cAMP-PKA, as demonstrated by the effects of both intracellular cAM P concentration and kinase inhibitor H89 on the Na+/H+ exchange activi ty. Based on these data, we proposed that betaNHE can exist in three d ifferent states (inactive I, activated A, and desensitized D). Convers ion of I to A needs the simultaneous phosphorylation by cAMP-PKA of si tes 1 and 2. These two sites might constitute the two neighboring cAMP -PKA sites located on the cytoplasmic loop as deduced from the oligonu cleotide sequence. Dephosphorylation of site 2 and subsequent binding of an arrestin-like protein are assumed to account for desensitization of the antiport. This putative role of arrestin is inferred from prev ious results demonstrating the presence in trout red cells (immunologi cal detection and cloning) of such a protein, which in addition has be en shown to bind to bovine rhodopsin as does retinal arrestin which pa rticipates in rhodopsin desensitization.