Chimaeras reveal the role of the catalytic core in the activation of the plasma membrane Ca2+ pump.
Isoform 2b of the plasma membrane calcium pump differs from the ubiquitous isoform 4b in the following: (a) higher basal activity in the absence of calmodulin; (b) higher affinity for calmodulin; and (c) higher affinity for Ca(2+) in the presence of calmodulin [Elwess, Filoteo, Enyedi and Penniston (1997) J. Biol. Chem. 272, 17981-17986]. To investigate which parts of the molecule determine these kinetic differences, we made four chimaeric constructs in which portions of isoform 2b were grafted into isoform 4b: chimaera I contains only the C-terminal regulatory region of isoform 2b; chimaera II contains the N-terminal moiety of isoform 2b, including both cytoplasmic loops; chimaera III contains the sequence of isoform 2b starting from the N-terminus to after the end of the first (small) cytoplasmic loop; and chimaera IV contains only the second (large) cytoplasmic loop. Surprisingly, chimaera I showed low basal activity in the absence of calmodulin and low affinity for calmodulin, unlike isoform 2b. In contrast, the chimaera containing both loops showed high basal activity, and Ca(2+) activation curves (both in the absence and in the presence of calmodulin) similar to those of isoform 2b. The rates of activation by calmodulin and of inactivation by calmodulin removal were measured, and the apparent K(d) for calmodulin was calculated from the ratio between these rate constants. The order of affinity was: 2b=II>4b=IV>III=I. From these results it is clear that the construct that most closely resembles isoform 2b is chimaera II. This shows that, in order to obtain an enzyme with properties similar to those of isoform 2b, both cytoplasmic loops are needed.
Ba-Thein
W
W
Department of Biochemistry and Molecular Biology, Mayo Foundation, 200 First Street South West, Rochester, MN 55905, USA.
Caride
A J
AJ
Enyedi
A
A
Pászty
K
K
Croy
C L
CL
Filoteo
A G
AG
Penniston
J T
JT
D000818:Animals; D001693:Biological Transport, Active / Q000187:drug effects; D019556:COS Cells; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000235:genetics / Q000378:metabolism*; D002147:Calmodulin / Q000494:pharmacology; D020134:Catalytic Domain; D027682:Cation Transport Proteins; D002462:Cell Membrane / Q000201:enzymology*; D053499:Plasma Membrane Calcium-Transporting ATPases; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D002147:Calmodulin; D027682:Cation Transport Proteins; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D053499:Plasma Membrane Calcium-Transporting ATPases; D000252:Calcium-Transporting ATPases; D002118:Calcium