A Signal for Endoplasmic Reticulum Retention Located at the Carboxyl Terminus of the Plasma Membrane Ca2+-ATPase Isoform 4CI

The plasma membrane Ca 2؉ -ATPase pump (PMCA) is an integral component of the Ca 2؉ signaling system which participates in signal transduction during agonist stimulated cell activation. To better understand the physiological function of the pump, isoform 1a (PMCA1a) was over-expressed in rat aortic endothelial cells using a stable transfection system under the control of a cytomegalovirus promoter. The cell lines selected after transfection with PMCA1a construct, expressed 3-4-fold increased pump protein which was mostly targeted to the plasma membrane as indicated by immunoperoxidase staining. Ca 2؉ uptake assays in a membrane preparation indicated a 3-4-fold increase in Ca 2؉ pumping activity in the transfected cells, and the expressed PMCA1a showed typical dependence on Ca ] i evoked by ATP and/or thapsigargin was lower but the plateau phase was similar in the PMCA1a expressing cells.

Section: Methodsmentioning

confidence: 99%

Consequences of Functional Expression of the Plasma Membrane Ca2+ Pump Isoform 1a

Liu

Fridman

et al. 1996

“…The other reagents were of the highest quality commercially available. The preparations of the recombinant viruses for PMCA2ACI, PMCA4CI, PMCA4CII, PMCA4⌬139, PMCA4⌬44, and PMCA1CI have already been described (11)(12)(13)(14)(15). An "empty" recombinant virus (control virus) was isolated during the preparation of the recombinant viruses.…”

Section: Methodsmentioning

confidence: 99%

“…The two truncated versions of the PMCA4CI pump, PMCA4CI⌬44 and PMCA4CI⌬139 (14) were also expressed (Fig. 1, A and B) to explore the possible involvement of the calmodulin binding region in the stimulation by ethanol.…”

Section: Effect Of Ethanol On Pmca Isoforms 29813mentioning

confidence: 99%

The Effect of Ethanol on the Plasma Membrane Calcium Pump Is Isoform-specific

Cervino¹,

Benaím²,

Carafoli³

et al. 1998

Self Cite

The effect of ethanol has been studied on four different isoforms of the plasma membrane Ca 2؉ -ATPase expressed in Sf9 cells with the help of the baculovirus system. The PMCA2CI protein was maximally activated by 0.5% ethanol, a concentration 8 -10 times lower than that needed to obtain the same effect on the PMCA4 protein or on the pump of erythrocyte membranes, which is a mixture of isoforms 1 and 4. Experiments performed with truncated pumps indicated that the stimulation by ethanol was lost if the C-terminal region between Lys 1065 and Lys 1161 , encompassing the calmodulin binding domain, was removed. These observations indicate that the stimulation is the result of a direct interaction of ethanol with the C-terminal regulatory domain of the Ca 2؉ pump.

“…COS-7 cells overexpressing the wild type hPMCA4CI pump ( Fig. 2A, WT) had a staining pattern typical of proteins targeted to the plasma membrane (23,36), i.e. diffuse fluorescence throughout the cell with well defined staining of the cell border.…”

Section: Membrane Targeting Of the Expressed Mutated Pmca4 Pumpmentioning

confidence: 99%

Single Amino Acid Mutations in Transmembrane Domain 5 Confer to the Plasma Membrane Ca2+ Pump Properties Typical of the Ca2+ Pump of Endo(sarco)plasmic Reticulum

Guerini¹,

Zecca-Mazza²,

Carafoli³

2000

Self Cite

Conserved residues in some of the transmembrane domains are proposed to mediate ion translocation by P-type pumps. The plasma membrane Ca 2؉ pump (PMCA) lacks 2 of these residues in transmembrane domains (TM) 5 and 8. In particular, a glutamic acid (Glu-771) residue in TM5, which is proposed to be involved in the binding and transport of Ca 2؉ by the sarcoplasmic reticulum Ca 2؉ pump (SERCA), is replaced by an alanine (Ala-854) in the PMCA pump. Ala-854 has been mutated to Glu, Asp, or Gln; Glu-975 in TM8, which is an Ala in the SERCA pump, has been mutated to Gln, Asp, or Ala. The mutants have been expressed in three cell systems, with or without the help of viruses. When expressed in large amounts in Sf9 cells, the mutated pumps were isolated and analyzed in the purified state. Two of the three TM8 mutants were correctly delivered to the plasma membrane and were active. All the TM5 mutants were retained in the endoplasmic reticulum; two of them (A854Q and A854E) retained activity. Their properties (La 3؉ sensitivity and decay of the phosphorylated intermediate, higher cooperativity of Ca 2؉ binding with a Hill's coefficient approaching 2) differed from those of the expressed wild type PMCA pump, and resembled those of the SERCA pump. Ca2ϩ transporting ATPases (Ca 2ϩ pumps) (1) remove Ca 2ϩ from the cytosol maintaining the low intracellular concentration necessary to its second messenger functions. The plasma membrane pump (PMCA) 1 shares structural (32% identity at the primary sequence level) and mechanistic properties with its intracellular counterpart (the sarco(endo)plasmic reticulum Ca 2ϩ ATPase or SERCA). As all P-type pumps, both ATPases form a high energy enzyme intermediate (phosphoenzyme) from ATP during the reaction cycle and are organized in the membrane with 10 putative transmembrane domains (TM) (2, 3). The second, and largest, cytosolic loop of both pumps contains the sites of phosphoenzyme formation and ATP binding (4), while regulatory regions like the calmodulin-binding domain are located at the C terminus of the PMCA enzyme (5, 6). Importantly, the inhibitor lanthanum acts differently on the two pumps; it stabilizes the phosphorylated intermediate of the PMCA pump, markedly increasing its steady state concentration (7). This effect, which is likely to reflect the inhibition of the conversion of the PMCA enzyme from the E 1 P to the E 2 P form (8), is not observed in the SERCA pump.The SERCA pump transports 2 calcium ions for each ATP hydrolyzed, while the PMCA pump only transports 1 (9 -11).