PMID- 9989931
OWN - NLM
STAT- MEDLINE
DCOM- 19990311
LR  - 20191210
IS  - 0003-9861 (Print)
IS  - 0003-9861 (Linking)
VI  - 362
IP  - 2
DP  - 1999 Feb 15
TI  - Effects of various amino acid 256 mutations on sarcoplasmic/endoplasmic reticulum 
      Ca2+ ATPase function and their role in the cellular adaptive response to 
      thapsigargin.
PG  - 225-32
AB  - Upon direct selection of mammalian cells for resistance to thapsigargin (TG), a 
      potent inhibitor of the sarcoplasmic/endoplasmic reticulum Ca2+ transport ATPase 
      (SERCA), the ATPase can acquire specific mutations at amino acid position 256 
      (aa256). In particular, Phe256 --> Leu and Phe256 --> Ser substitutions can occur 
      upon TG selection, with each substitution resulting in a SERCA that is 4- to 
      5-fold resistant to TG inhibition (M. Yu et al., J. Biol. Chem. 273, 3542-3546, 
      1998). We have now identified a third substitution, i.e., Phe256 --> Val, that 
      occurs when the Chinese hamster lung fibroblast cell line DC-3F is selected for 
      TG resistance. Although the Phe256 --> Val substitution at codon 256 results in a 
      SERCA whose enzymological properties in terms of Ca2+ transport and ATP 
      hydrolysis are essentially similar to that of wild-type (wt) SERCA, the mutant 
      enzyme is more than 40-fold resistant to TG inhibition. To analyze further the 
      role of aa256 in TG-SERCA interactions, mutational analysis of this particular 
      residue was also carried out. Of all the mutations introduced, only the Phe256 
      --> Glu substitution interferes with expression of the ATPase. The Phe256 --> Arg 
      substitution does not interfere with SERCA expression, but the resulting enzyme 
      is totally inactive. In terms of sensitivity of the various mutants to TG, 
      maximal reduction in the ATPase's affinity for TG occurs with amino acid 
      substitutions containing branched side chains, i.e. with the Phe256 --> Val, 
      Phe256 --> Ile, and Phe256 --> Thr mutants. Since a corresponding Phe is 
      conserved in the Na+, K+-ATPase which is not sensitive to TG, our findings 
      suggest that this amino acid provides stabilization of the stalk segment with 
      respect to the membrane interface, thereby optimizing specific interactions of TG 
      with neighboring S3 residues (L. Zhong and G. Inesi, J. Biol. Chem. 273, 
      12994-12998, 1998). It is likely that a relatively high frequency of codon 256 
      mutations favor the aa256 mutants as a specific adaptive response to TG 
      selection.
CI  - Copyright 1999 Academic Press.
FAU - Yu, M
AU  - Yu M
AD  - Department of Medicine, Greenebaum Cancer Center, Baltimore, Maryland, 21201, 
      USA.
FAU - Lin, J
AU  - Lin J
FAU - Khadeer, M
AU  - Khadeer M
FAU - Yeh, Y
AU  - Yeh Y
FAU - Inesi, G
AU  - Inesi G
FAU - Hussain, A
AU  - Hussain A
LA  - eng
GR  - P01HL27867/HL/NHLBI NIH HHS/United States
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PT  - Research Support, U.S. Gov't, Non-P.H.S.
PT  - Research Support, U.S. Gov't, P.H.S.
PL  - United States
TA  - Arch Biochem Biophys
JT  - Archives of biochemistry and biophysics
JID - 0372430
RN  - 0 (ATP Binding Cassette Transporter, Subfamily B)
RN  - 0 (Recombinant Proteins)
RN  - 67526-95-8 (Thapsigargin)
RN  - 83HN0GTJ6D (Cyclosporine)
RN  - 8L70Q75FXE (Adenosine Triphosphate)
RN  - EC 7.2.2.10 (Calcium-Transporting ATPases)
RN  - SY7Q814VUP (Calcium)
SB  - IM
MH  - ATP Binding Cassette Transporter, Subfamily B/antagonists & inhibitors/metabolism
MH  - Adaptation, Physiological/*drug effects
MH  - Adenosine Triphosphate/metabolism
MH  - Amino Acid Substitution/drug effects/genetics/*physiology
MH  - Animals
MH  - Base Sequence
MH  - Blotting, Western
MH  - COS Cells
MH  - Calcium/metabolism
MH  - Calcium-Transporting ATPases/antagonists & inhibitors/genetics/*metabolism
MH  - Cell Line
MH  - Cricetinae
MH  - Cyclosporine/pharmacology
MH  - Drug Resistance
MH  - Fibroblasts
MH  - Lung
MH  - Microsomes/enzymology/metabolism
MH  - Recombinant Proteins/metabolism
MH  - Sarcoplasmic Reticulum/drug effects/*enzymology/metabolism
MH  - Thapsigargin/metabolism/*pharmacology
EDAT- 1999/02/17 00:00
MHDA- 1999/02/17 00:01
CRDT- 1999/02/17 00:00
PHST- 1999/02/17 00:00 [pubmed]
PHST- 1999/02/17 00:01 [medline]
PHST- 1999/02/17 00:00 [entrez]
AID - S0003-9861(98)91049-9 [pii]
AID - 10.1006/abbi.1998.1049 [doi]
PST - ppublish
SO  - Arch Biochem Biophys. 1999 Feb 15;362(2):225-32. doi: 10.1006/abbi.1998.1049.