Co-reconstitution of phospholamban mutants with the Ca-ATPase reveals dependence of inhibitory function on phospholamban structure

Laxma G. Reddy, Joseph M. Autry, Larry Jones, David D. Thomas

Research output: Contribution to journalArticle

49 Citations (Scopus)

Abstract

Phospholamban (PLB), a 52-amino acid integral membrane protein, regulates the Ca-ATPase (calcium pump) in cardiac sarcoplasmic reticulum through PLB phosphorylation mediated by β-adrenergic stimulation. Based on site-directed mutagenesis and coexpression with Ca-ATPase (SERCA2a) in Sf21 insect cells or in HEK 293 cells, and on spin label detection of PLB oligomeric state in lipid bilayers, it has been proposed that the monomeric form of PLB is the inhibitory species, and depolymerization of PLB is essential for its regulatory function. Here we have studied the relationship between PLB oligomeric state and function by in vitro co-reconstitution of PLB and its mutants with purified Ca-ATPase. We compared wild type-PLB (wt- PLB), which is primarily a pentamer on SDS-polyacrylamide gel electrophoresis (PAGE) at 25°C, with two of its mutants, C41L-PLB and L37A-PLB, that are primarily tetramer and monomer, respectively. We found that the monomeric mutant L37A-PLB is a more potent inhibitor than wt-PLB, supporting the previous proposal that PLB monomer is the inhibitory species. On the other hand, C41L-PLB, which has a monomeric fraction comparable to that of wt-PLB on SDS-PAGE at 25°C, has no inhibitory activity when assayed at 25°C. However, at 37°C, a 3-fold increase in the monomeric fraction of C41L-PLB on SDS-PAGE resulted in inhibitory activity comparable to that of wt-PLB. Upon increasing the temperature from 25 to 37°C, no change in fraction monomer or inhibitory activity for wt-PLB and L37A-PLB was observed. Based on these results, the extent of inhibition of Ca-ATPase by PLB or its mutants appears to depend not only on the propensity of PLB to dissociate into monomers but also on the relative potency of the particular PLB monomer when interacting with the Ca-ATPase.

Original languageEnglish
Pages (from-to)7649-7655
Number of pages7
JournalJournal of Biological Chemistry
Volume274
Issue number12
DOIs
StatePublished - Mar 19 1999

Fingerprint

Adenosine Triphosphatases
Monomers
phospholamban
Electrophoresis
Polyacrylamide Gel Electrophoresis
Sf9 Cells
Spin Labels
Mutagenesis
Depolymerization
Phosphorylation
Lipid bilayers
HEK293 Cells
Lipid Bilayers
Sarcoplasmic Reticulum
Site-Directed Mutagenesis

ASJC Scopus subject areas

  • Biochemistry

Cite this

Co-reconstitution of phospholamban mutants with the Ca-ATPase reveals dependence of inhibitory function on phospholamban structure. / Reddy, Laxma G.; Autry, Joseph M.; Jones, Larry; Thomas, David D.

In: Journal of Biological Chemistry, Vol. 274, No. 12, 19.03.1999, p. 7649-7655.

Research output: Contribution to journalArticle

@article{f4522bf4db72408385a928bd6945aef9,
title = "Co-reconstitution of phospholamban mutants with the Ca-ATPase reveals dependence of inhibitory function on phospholamban structure",
abstract = "Phospholamban (PLB), a 52-amino acid integral membrane protein, regulates the Ca-ATPase (calcium pump) in cardiac sarcoplasmic reticulum through PLB phosphorylation mediated by β-adrenergic stimulation. Based on site-directed mutagenesis and coexpression with Ca-ATPase (SERCA2a) in Sf21 insect cells or in HEK 293 cells, and on spin label detection of PLB oligomeric state in lipid bilayers, it has been proposed that the monomeric form of PLB is the inhibitory species, and depolymerization of PLB is essential for its regulatory function. Here we have studied the relationship between PLB oligomeric state and function by in vitro co-reconstitution of PLB and its mutants with purified Ca-ATPase. We compared wild type-PLB (wt- PLB), which is primarily a pentamer on SDS-polyacrylamide gel electrophoresis (PAGE) at 25°C, with two of its mutants, C41L-PLB and L37A-PLB, that are primarily tetramer and monomer, respectively. We found that the monomeric mutant L37A-PLB is a more potent inhibitor than wt-PLB, supporting the previous proposal that PLB monomer is the inhibitory species. On the other hand, C41L-PLB, which has a monomeric fraction comparable to that of wt-PLB on SDS-PAGE at 25°C, has no inhibitory activity when assayed at 25°C. However, at 37°C, a 3-fold increase in the monomeric fraction of C41L-PLB on SDS-PAGE resulted in inhibitory activity comparable to that of wt-PLB. Upon increasing the temperature from 25 to 37°C, no change in fraction monomer or inhibitory activity for wt-PLB and L37A-PLB was observed. Based on these results, the extent of inhibition of Ca-ATPase by PLB or its mutants appears to depend not only on the propensity of PLB to dissociate into monomers but also on the relative potency of the particular PLB monomer when interacting with the Ca-ATPase.",
author = "Reddy, {Laxma G.} and Autry, {Joseph M.} and Larry Jones and Thomas, {David D.}",
year = "1999",
month = "3",
day = "19",
doi = "10.1074/jbc.274.12.7649",
language = "English",
volume = "274",
pages = "7649--7655",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "12",

}

TY - JOUR

T1 - Co-reconstitution of phospholamban mutants with the Ca-ATPase reveals dependence of inhibitory function on phospholamban structure

AU - Reddy, Laxma G.

AU - Autry, Joseph M.

AU - Jones, Larry

AU - Thomas, David D.

PY - 1999/3/19

Y1 - 1999/3/19

N2 - Phospholamban (PLB), a 52-amino acid integral membrane protein, regulates the Ca-ATPase (calcium pump) in cardiac sarcoplasmic reticulum through PLB phosphorylation mediated by β-adrenergic stimulation. Based on site-directed mutagenesis and coexpression with Ca-ATPase (SERCA2a) in Sf21 insect cells or in HEK 293 cells, and on spin label detection of PLB oligomeric state in lipid bilayers, it has been proposed that the monomeric form of PLB is the inhibitory species, and depolymerization of PLB is essential for its regulatory function. Here we have studied the relationship between PLB oligomeric state and function by in vitro co-reconstitution of PLB and its mutants with purified Ca-ATPase. We compared wild type-PLB (wt- PLB), which is primarily a pentamer on SDS-polyacrylamide gel electrophoresis (PAGE) at 25°C, with two of its mutants, C41L-PLB and L37A-PLB, that are primarily tetramer and monomer, respectively. We found that the monomeric mutant L37A-PLB is a more potent inhibitor than wt-PLB, supporting the previous proposal that PLB monomer is the inhibitory species. On the other hand, C41L-PLB, which has a monomeric fraction comparable to that of wt-PLB on SDS-PAGE at 25°C, has no inhibitory activity when assayed at 25°C. However, at 37°C, a 3-fold increase in the monomeric fraction of C41L-PLB on SDS-PAGE resulted in inhibitory activity comparable to that of wt-PLB. Upon increasing the temperature from 25 to 37°C, no change in fraction monomer or inhibitory activity for wt-PLB and L37A-PLB was observed. Based on these results, the extent of inhibition of Ca-ATPase by PLB or its mutants appears to depend not only on the propensity of PLB to dissociate into monomers but also on the relative potency of the particular PLB monomer when interacting with the Ca-ATPase.

AB - Phospholamban (PLB), a 52-amino acid integral membrane protein, regulates the Ca-ATPase (calcium pump) in cardiac sarcoplasmic reticulum through PLB phosphorylation mediated by β-adrenergic stimulation. Based on site-directed mutagenesis and coexpression with Ca-ATPase (SERCA2a) in Sf21 insect cells or in HEK 293 cells, and on spin label detection of PLB oligomeric state in lipid bilayers, it has been proposed that the monomeric form of PLB is the inhibitory species, and depolymerization of PLB is essential for its regulatory function. Here we have studied the relationship between PLB oligomeric state and function by in vitro co-reconstitution of PLB and its mutants with purified Ca-ATPase. We compared wild type-PLB (wt- PLB), which is primarily a pentamer on SDS-polyacrylamide gel electrophoresis (PAGE) at 25°C, with two of its mutants, C41L-PLB and L37A-PLB, that are primarily tetramer and monomer, respectively. We found that the monomeric mutant L37A-PLB is a more potent inhibitor than wt-PLB, supporting the previous proposal that PLB monomer is the inhibitory species. On the other hand, C41L-PLB, which has a monomeric fraction comparable to that of wt-PLB on SDS-PAGE at 25°C, has no inhibitory activity when assayed at 25°C. However, at 37°C, a 3-fold increase in the monomeric fraction of C41L-PLB on SDS-PAGE resulted in inhibitory activity comparable to that of wt-PLB. Upon increasing the temperature from 25 to 37°C, no change in fraction monomer or inhibitory activity for wt-PLB and L37A-PLB was observed. Based on these results, the extent of inhibition of Ca-ATPase by PLB or its mutants appears to depend not only on the propensity of PLB to dissociate into monomers but also on the relative potency of the particular PLB monomer when interacting with the Ca-ATPase.

UR - http://www.scopus.com/inward/record.url?scp=0033583036&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0033583036&partnerID=8YFLogxK

U2 - 10.1074/jbc.274.12.7649

DO - 10.1074/jbc.274.12.7649

M3 - Article

C2 - 10075652

AN - SCOPUS:0033583036

VL - 274

SP - 7649

EP - 7655

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 12

ER -