Abstract
High levels of resistance to thapsigargin (TG), a specific inhibitor of intracellular Ca2+ transport ATPases (SERCAs), can be developed in culture by stepwise exposure of mammalian cells to increasing concentrations of TG. We have identified, in two independently selected TG-resistant hamster cell lines of different lineages, mutant forms of SERCA. In the TG-resistant Chinese hamster lung fibroblast cell line DC-3F/TG, a T --> C change at nucleotide 766 introduces a Phe256 --> Leu alteration within the first cytosolic loop of the SERCA. In contrast, in the TG-resistant Syrian hamster smooth muscle cell line DDT/TG 4 microM, a T --> C change at nucleotide 767 introduces a Phe256 --> Ser mutation at that position. When these specific mutations are introduced into a wild-type full-length avian SERCA1 cDNA, transfection experiments reveal that Ca2+ transport function and ATP hydrolytic activity are not altered by such mutations. However, a 4-5-fold resistance to TG inhibition of Ca2+ transport function occurs upon the introduction of either the Phe256 --> Leu or the Phe256 --> Ser mutation into wild-type SERCA1. These specific mutations also render the hydrolytic activity of the ATPase resistant to inhibition by TG. Our results not only implicate amino acid 256 in TG-SERCA interactions, but also demonstrate that specific mutations within SERCA can mediate resistance to TG.
Highlights
From the ‡Division of Oncology, Department of Medicine, Greenebaum Cancer Center and §Department of Biological Chemistry, School of Medicine, University of Maryland, Baltimore, Maryland 21201, and the ¶Baltimore Veterans Affairs Medical Center, Baltimore, Maryland 21201
In the TG-resistant Syrian hamster smooth muscle cell line DDT/TG 4M, a T 3 C change at nucleotide 767 introduces a Phe256 3 Ser mutation at that position. When these specific mutations are introduced into a wild-type full-length avian SERCA1 cDNA, transfection experiments reveal that Ca2؉ transport function and ATP hydrolytic activity are not altered by such mutations
This is the first demonstration of naturally occurring mutations within SERCA during development of the TG-resistant phenotype, and our results suggest that Phe256, which lies within the first cytosolic loop just upstream of the M3 domain, represents a “ hot spot” for mutations upon TG selection, and is potentially involved in TG-SERCA interactions
Summary
(Received for publication, September 16, 1997, and in revised form, November 18, 1997). In the TG-resistant Syrian hamster smooth muscle cell line DDT/TG 4M, a T 3 C change at nucleotide 767 introduces a Phe256 Ser mutation at that position When these specific mutations are introduced into a wild-type full-length avian SERCA1 cDNA, transfection experiments reveal that Ca2؉ transport function and ATP hydrolytic activity are not altered by such mutations. A 4 –5-fold resistance to TG inhibition of Ca2؉ transport function occurs upon the introduction of either the Phe256 Leu or the Phe256 Ser mutation into wild-type SERCA1 These specific mutations render the hydrolytic activity of the ATPase resistant to inhibition by TG. When either of these two specific mutations are introduced into wild-type (wt) SERCA1, both Ca2ϩ transport and ATP hydrolytic activities of the resulting ATPase become resistant to inhibition by TG. This is the first demonstration of naturally occurring mutations within SERCA during development of the TG-resistant phenotype, and our results suggest that Phe256, which lies within the first cytosolic loop just upstream of the M3 domain, represents a “ hot spot” for mutations upon TG selection, and is potentially involved in TG-SERCA interactions
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