Functional expression of the human growth factor activatable Na+/H+ antiporter (NHE-1) in baculovirus-infected cells.
We constructed a recombinant baculovirus, based on Autographa californica nuclear polyhedrosis virus, containing the human Na+/H+ antiporter cDNA under control of the polyhedrin promoter. When infected with this recombinant baculovirus, the Sf9 cell line, derived from Spodoptera frugiperda, expresses a fully functional Na+/H+ antiporter as measured by the generation of an amiloride-sensitive Na+ influx in response to an acid load. The Na+/H(+)-exchange activity, not detectable in Sf9 cells, emerges 18 h after infection and continues to increase over the next two days to reach a maximal value about 20-fold higher than in cultured mammalian fibroblasts. Parallel to this activity, infected cells express a single immunoreactive polypeptide of 85 kDa that represents a non-glycosylated form of the 110-kDa mature human antiporter. We estimated that only 10% of the expressed protein is in a functional state. Not only is the antiporter expressed in insect cells phosphorylated, but also, like in mammalian cells, phosphorylation is increased in response to phorbol esters and okadaic acid. Moreover, tumor promoters apparently modify the same antiporter site in both insect and mammalian cells. We conclude that, with this high level of functional expression and apparently conserved signaling machinery, the present system opens the way to the biochemistry of the transporter including identification of the growth factor stimulated phosphorylation sites.
Fafournoux
P
P
Centre de Biochimie-CNRS, Université de Nice, France.
Ghysdael
J
J
Sardet
C
C
Pouysségur
J
J
D000818:Animals; D016367:Baculoviridae; D001692:Biological Transport; D002352:Carrier Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D005822:Genetic Vectors; D013006:Growth Hormone / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D009036:Moths; D010449:Peptide Mapping; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011233:Precipitin Tests; D011994:Recombinant Proteins / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers
D002352:Carrier Proteins; D010750:Phosphoproteins; D011994:Recombinant Proteins; D017923:Sodium-Hydrogen Exchangers; D013006:Growth Hormone; D012964:Sodium