Abstract
Alternate splicing of human plasma membrane calcium pump isoform 4 (hPMCA4) transcripts causes the expression of two variants, hPMCA4a and hPMCA4b, which have different downstream regulatory regions. Of the two, hPMCA4a has a lower affinity for calmodulin and a lower effective affinity for Ca2+ (Enyedi, A., Verma, A. K., Heim, R., Adamo, H. P., Filoteo, A. G., Strehler, E. E., and Penniston, J. T. (1994) J. Biol. Chem. 269, 41-43). Additional consequences of the alternate splice were studied by analyzing the characteristics of constructs (expressed in COS-1 cells) containing different portions of the carboxyl terminus of hPMCA4a. Our results show striking differences in the structure of the calmodulin-binding and autoinhibitory domains of the two variants. The calmodulin-binding region of hPMCA4b is a region of about 28 residues, whereas that of hPMCA4a is about 49 residues long and is probably interrupted by a region not involved in the binding. The autoinhibitory region of hPMCA4b (a part of the downstream region that keeps the molecule inactive in the absence of Ca2+-calmodulin) is divided between the 28-residue calmodulin-binding region and a downstream region, whereas in hPMCA4a, all of it is contained within the 49-residue calmodulin-binding region.
Highlights
Responsible for calmodulin binding was shown to bind calmodulin even more tightly than the intact enzyme (Enyedi et al, 1989)
Adding back all 28 residues of the calmodulin-binding domain, which made a construct called ct92 (Verma et al, 1994; see Fig. 1), induced a substantial inhibition of the pump in the absence of calmodulin. This inhibition, was only about 2⁄3 of the inhibition that was observed in the full-length pump, suggesting that other segments of the downstream region are involved in selfinhibition of isoform 4b. ct92, on the other hand, had an apparent affinity for calmodulin just as high as that of the fulllength enzyme, indicating that the portion of hPMCA4b downstream of the 28-residue calmodulin-binding domain played no role in calmodulin binding
The upstream 19 residues of the calmodulin-binding domain are conserved in all the PMCA isoforms, but an alternate RNA splice affecting the middle of the sequence coding for this region changes the structure of the rest of the calmodulin-binding domain and the entire carboxyl terminus
Summary
Responsible for calmodulin binding was shown to bind calmodulin even more tightly than the intact enzyme (Enyedi et al, 1989) This peptide inhibited the pump that was activated by proteolytic removal of the calmodulin-binding domain and the whole carboxyl terminus. Closer inspection of 4a(ct56), showed that unlike 4b(ct92), it has lower affinity for calmodulin than its full-length parent and that like 4b(ct92) its activity is higher in the absence of calmodulin than the activity of its parent To further study this region, more residues of the carboxyl terminus were added to 4a(ct56) and two additional constructs, called 4a(ct44) and 4a(ct35) were produced. The characteristics of 4a(ct35) appeared to be identical to those of full-length hPMCA4a, indicating that in hPMCA4a both calmodulin-binding and inhibitory characteristics reside in a region of about 49 residues at the carboxyl terminus
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