The cytoplasmic domain of the Na+/H+ exchangers (NHEs) dictates the nature of the hormonal response: behavior of a chimeric human NHE1/trout beta NHE antiporter.
Studies of the effect of cAMP on the cloned Na+/H+ exchangers (NHEs) are difficult to interpret as variable results have been reported for the different isoforms when expressed in various cell types. We took advantage of the fact that the human NHE1 and the trout erythrocyte beta NHE, when expressed in the same cell line, PS120, respond differently to cAMP (NHE1 is insensitive, beta NHE is activated) to analyze the molecular mechanisms of cAMP activation. We constructed both a chimera between NHE1 and beta NHE and a set of beta NHE mutants to delineate the critical parts of the molecule involved in the activation process. NHE1 becomes cAMP stimulated when its cytoplasmic domain is replaced by the cytoplasmic domain of beta NHE; thus, the cytoplasmic C terminus of beta NHE, which contains two cAMP-dependent consensus sequences, is essential to confer cAMP dependence. Serine to glycine substitution of only one of the two protein kinase A (PKA) consensus sites decreased by 60% the ability of cAMP to activate Na+/H+ exchange. Simultaneous Ser to Gly substitution of the two PKA consensus sites decreased the cAMP-mediated activation by 72%. The residual activation required a cytoplasmic fragment (aa 559-661) that contains four sequences considered likely as putative PKA consensus sites. The results obtained with the chimeric NHE also demonstrated that if the cytoplasmic C terminus is crucially involved in the hormonal activation, the rate of Na+/H+ exchange so induced can be modulated by the nature of the interaction between the N- and C-terminal domains.
Borgese
F
F
Laboratoire Jean Maetz, Départment de Biologie Cellulaire et Moléculaire du Commissariat à l'Energie Atomique, Villefranche-sur-Mer, France.
Malapert
M
M
Fievet
B
B
Pouyssegur
J
J
Motais
R
R
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D000242:Cyclic AMP / Q000502:physiology; D003593:Cytoplasm / Q000737:chemistry; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry; D008969:Molecular Sequence Data; D010703:Phorbol Esters / Q000494:pharmacology; D011993:Recombinant Fusion Proteins; D017923:Sodium-Hydrogen Exchangers / Q000737:chemistry*; D013329:Structure-Activity Relationship; D014337:Trout
D008565:Membrane Proteins; D010703:Phorbol Esters; D011993:Recombinant Fusion Proteins; D017923:Sodium-Hydrogen Exchangers; D000242:Cyclic AMP