Abstract
The role of ATP-dependent calcium uptake into intracellular storage compartments is an essential feature of hormonally induced calcium signaling. Thapsigargin, a non-phorboid tumor promoter, increasingly is being used to manipulate calcium stores because it induces a hormone-like elevation of cytosolic calcium. It has been suggested that thapsigargin acts through inhibition of the endoplasmic reticulum calcium pump. We have directly tested the specificity of thapsigargin on all of the known intracellular-type calcium pumps (referred to as the sarcoplasmic or endoplasmic reticulum Ca-ATPase family (SERCA]. Full-length cDNA clones encoding SERCA1, SERCA2a, SERCA2b, and SERCA3 enzymes were expressed in COS cells, and both calcium uptake and calcium-dependent ATPase activity were assayed in microsomes isolated from them. Thapsigargin inhibited all of the SERCA isozymes with equal potency. Furthermore, similar doses of thapsigargin abolished the calcium uptake and ATPase activity of sarcoplasmic reticulum isolated from fast twitch and cardiac muscle but had no influence on either the plasma membrane Ca-ATPase or Na,K-ATPase. The interaction of thapsigargin with the SERCA isoforms is rapid, stoichiometric, and essentially irreversible. These properties demonstrate that thapsigargin interacts with a recognition site found in, and only in, all members of the endoplasmic and sarcoplasmic reticulum calcium pump family.
Highlights
The role of ATP-dependent calcium uptake into in- reported evidence for the existence of two distinct tracellular storage compartments is an essential fea- calcium storage compartments, which under certain circumture of hormonally induced calcium signaling
We have directly tested the specificity of thapsigargin on all of the known intracellular-type calcium pumps (referred to as the sarcoplasmic or endoplasmic reticulum Ca-ATPase family (SERCA)).Full-length cDNA clones encoding SERCA1, SERCA2a, SERCASb, and SERCA3 enzymes were expressed in COS cells, and both calcium uptake and calcium-dependent ATPase stances may be in communication (Iinoet al., 1988; Ghosh et al, 1989; Thevenod et al, 1989)
We refer to these related species of pumps as the sarcoplasmicor endoplasmic activitywere assayed in microsomes isolated from reticulum calcium ATPase family (SERCA).Theyareenthem
Summary
K:) 1991 by The American Society for Biochemistry and Molecular Biology, Inc. Vol 266,No 26, Issue of September 15, PP. 17067-17071, 1991 Printed in U.S.A. The interaction of thapsigargin with the SERCA isoforms is rapid, stoichiometric, and essentially irreversible These properties demonstrate that thapsigargin interacts with a recognition site found in, and only in, all members of the endoplasmic and sarcoplasmic reticulum calcium pump family. Thapsigargin, a plant-derivedsesquiterpenelactone,has beenidentified as a non-phorboidtumorpromoter whose mode of action appears to resultfrom the emptying of intracellular calcium stores (Thastrup,1990), as a consequence of inhibiting the uptake pathway (Thastrup et al, 1990) For this reason, thapsigargin hasrecently found much popularity as an agent both to define and to manipulate intracellular calcium pools (Bian et al, 1991; Foskett et al, 1991; Verma et al, 1990; Kwan et al, 1990). Inhibition of requiring coordinated cycles of emptying and refilling (Ber- only an endoplasmic reticulum calcium pump suggests that ridge, 1990; Meyer,1991).,several laboratories have thetarget for thapsigarginmightbetheunique carboxylterminal tail of the SERCA2b protein or possibly a unique.
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