Regulation of G(s) α activity in the myocardium: Effects of chronic pressure overload

C. Homcy, Dorothy Vatner, Stephen Vatner, J. P. Longabaugh, B. Nadal-Ginard

Research output: Contribution to journalArticle

Abstract

Alterations in the level and function of the stimulatory guanyl nucleotide binding protein (G(s)) from the cardiac sarcolemma were examined in a canine model of heart failure. Previous investigations have demonstrated both a loss of β-adrenergic agonist high-affinity binding sites and a decreased adenylate cyclase activity in sarcolemma from failing hearts. Using cholera toxin and [32P]NAD, we labelled the alpha subunit of G(s) (G(s)α) and found a 59% reduction in the level of this protein. Further, a 50% reduction in G(s) activity was noted in a reconstitution assay utilizing membranes from the mouse S49 lymphoma cell line cyc-, which is deficient in G(s). These data suggest that, in this model of pressure-overload left ventricular failure, the acquired defect in the β-adrenergic receptor/adenylate cyclase system involves a deficiency in the coupling protein G. Based on these findings, we set out to characterize the expression of the G(s) α subunit in the heart. Three cDNAs from a cardiac λgt10 library were completely sequenced and two clones (6A and 7A), both 2kb in length, were found to differ by only one codon (AGT) in their translated region resulting in the insertion of an additional serine residue after amino acid position 71 in cDNA 7A. In their untranslated regions, these 2 clones employed different polyadenylation sites. The third clone (cDNA 6B), 1.5kb in length but lacking a complete 5' end, differed from 6A over a 46 nucleotide stretch which also began at residue 71. Although cDNAs containing this 46 nucleotide splice have been reported to encode for the larger MW form of G(s) α, this protein species was not detectable in canine sarcolemma, a finding possibly related to the fact that cDNA 6B contained a novel sequence 5' to this position as well. Based on the sequences of these three cDNAs as well as S1 nuclease analyses, a minimum of two alternatively spliced exons in addition to duplicated acceptor splice sites need to be involved in the generation of their corresponding mRNA species. Southern blots of genomic DNA were consistent with the existence of a single copy of the G(s) α gene. These findings suggests that the activity of G(s) α is regulated via the expression of multiple isoforms generated via alternative splicing of a single copy gene. Studies are now aimed at characterizing the expression of G(s) α in the hearts of animals with pressure-overload LV hypertrophy and failure.

Original languageEnglish (US)
JournalJournal of Molecular and Cellular Cardiology
Volume20
Issue numberSUPPL. 1
StatePublished - Jan 1 1988
Externally publishedYes

Fingerprint

Myocardium
Complementary DNA
Pressure
Sarcolemma
Nucleotides
Clone Cells
Adenylyl Cyclases
Canidae
Gs GTP-Binding Protein alpha Subunits
Untranslated Regions
Adrenergic Agonists
RNA Splice Sites
Polyadenylation
Proteins
Cholera Toxin
Alternative Splicing
Southern Blotting
Codon
NAD
Adrenergic Receptors

All Science Journal Classification (ASJC) codes

  • Cardiology and Cardiovascular Medicine
  • Molecular Biology

Cite this

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title = "Regulation of G(s) α activity in the myocardium: Effects of chronic pressure overload",
abstract = "Alterations in the level and function of the stimulatory guanyl nucleotide binding protein (G(s)) from the cardiac sarcolemma were examined in a canine model of heart failure. Previous investigations have demonstrated both a loss of β-adrenergic agonist high-affinity binding sites and a decreased adenylate cyclase activity in sarcolemma from failing hearts. Using cholera toxin and [32P]NAD, we labelled the alpha subunit of G(s) (G(s)α) and found a 59{\%} reduction in the level of this protein. Further, a 50{\%} reduction in G(s) activity was noted in a reconstitution assay utilizing membranes from the mouse S49 lymphoma cell line cyc-, which is deficient in G(s). These data suggest that, in this model of pressure-overload left ventricular failure, the acquired defect in the β-adrenergic receptor/adenylate cyclase system involves a deficiency in the coupling protein G. Based on these findings, we set out to characterize the expression of the G(s) α subunit in the heart. Three cDNAs from a cardiac λgt10 library were completely sequenced and two clones (6A and 7A), both 2kb in length, were found to differ by only one codon (AGT) in their translated region resulting in the insertion of an additional serine residue after amino acid position 71 in cDNA 7A. In their untranslated regions, these 2 clones employed different polyadenylation sites. The third clone (cDNA 6B), 1.5kb in length but lacking a complete 5' end, differed from 6A over a 46 nucleotide stretch which also began at residue 71. Although cDNAs containing this 46 nucleotide splice have been reported to encode for the larger MW form of G(s) α, this protein species was not detectable in canine sarcolemma, a finding possibly related to the fact that cDNA 6B contained a novel sequence 5' to this position as well. Based on the sequences of these three cDNAs as well as S1 nuclease analyses, a minimum of two alternatively spliced exons in addition to duplicated acceptor splice sites need to be involved in the generation of their corresponding mRNA species. Southern blots of genomic DNA were consistent with the existence of a single copy of the G(s) α gene. These findings suggests that the activity of G(s) α is regulated via the expression of multiple isoforms generated via alternative splicing of a single copy gene. Studies are now aimed at characterizing the expression of G(s) α in the hearts of animals with pressure-overload LV hypertrophy and failure.",
author = "C. Homcy and Dorothy Vatner and Stephen Vatner and Longabaugh, {J. P.} and B. Nadal-Ginard",
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Regulation of G(s) α activity in the myocardium : Effects of chronic pressure overload. / Homcy, C.; Vatner, Dorothy; Vatner, Stephen; Longabaugh, J. P.; Nadal-Ginard, B.

In: Journal of Molecular and Cellular Cardiology, Vol. 20, No. SUPPL. 1, 01.01.1988.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Regulation of G(s) α activity in the myocardium

T2 - Effects of chronic pressure overload

AU - Homcy, C.

AU - Vatner, Dorothy

AU - Vatner, Stephen

AU - Longabaugh, J. P.

AU - Nadal-Ginard, B.

PY - 1988/1/1

Y1 - 1988/1/1

N2 - Alterations in the level and function of the stimulatory guanyl nucleotide binding protein (G(s)) from the cardiac sarcolemma were examined in a canine model of heart failure. Previous investigations have demonstrated both a loss of β-adrenergic agonist high-affinity binding sites and a decreased adenylate cyclase activity in sarcolemma from failing hearts. Using cholera toxin and [32P]NAD, we labelled the alpha subunit of G(s) (G(s)α) and found a 59% reduction in the level of this protein. Further, a 50% reduction in G(s) activity was noted in a reconstitution assay utilizing membranes from the mouse S49 lymphoma cell line cyc-, which is deficient in G(s). These data suggest that, in this model of pressure-overload left ventricular failure, the acquired defect in the β-adrenergic receptor/adenylate cyclase system involves a deficiency in the coupling protein G. Based on these findings, we set out to characterize the expression of the G(s) α subunit in the heart. Three cDNAs from a cardiac λgt10 library were completely sequenced and two clones (6A and 7A), both 2kb in length, were found to differ by only one codon (AGT) in their translated region resulting in the insertion of an additional serine residue after amino acid position 71 in cDNA 7A. In their untranslated regions, these 2 clones employed different polyadenylation sites. The third clone (cDNA 6B), 1.5kb in length but lacking a complete 5' end, differed from 6A over a 46 nucleotide stretch which also began at residue 71. Although cDNAs containing this 46 nucleotide splice have been reported to encode for the larger MW form of G(s) α, this protein species was not detectable in canine sarcolemma, a finding possibly related to the fact that cDNA 6B contained a novel sequence 5' to this position as well. Based on the sequences of these three cDNAs as well as S1 nuclease analyses, a minimum of two alternatively spliced exons in addition to duplicated acceptor splice sites need to be involved in the generation of their corresponding mRNA species. Southern blots of genomic DNA were consistent with the existence of a single copy of the G(s) α gene. These findings suggests that the activity of G(s) α is regulated via the expression of multiple isoforms generated via alternative splicing of a single copy gene. Studies are now aimed at characterizing the expression of G(s) α in the hearts of animals with pressure-overload LV hypertrophy and failure.

AB - Alterations in the level and function of the stimulatory guanyl nucleotide binding protein (G(s)) from the cardiac sarcolemma were examined in a canine model of heart failure. Previous investigations have demonstrated both a loss of β-adrenergic agonist high-affinity binding sites and a decreased adenylate cyclase activity in sarcolemma from failing hearts. Using cholera toxin and [32P]NAD, we labelled the alpha subunit of G(s) (G(s)α) and found a 59% reduction in the level of this protein. Further, a 50% reduction in G(s) activity was noted in a reconstitution assay utilizing membranes from the mouse S49 lymphoma cell line cyc-, which is deficient in G(s). These data suggest that, in this model of pressure-overload left ventricular failure, the acquired defect in the β-adrenergic receptor/adenylate cyclase system involves a deficiency in the coupling protein G. Based on these findings, we set out to characterize the expression of the G(s) α subunit in the heart. Three cDNAs from a cardiac λgt10 library were completely sequenced and two clones (6A and 7A), both 2kb in length, were found to differ by only one codon (AGT) in their translated region resulting in the insertion of an additional serine residue after amino acid position 71 in cDNA 7A. In their untranslated regions, these 2 clones employed different polyadenylation sites. The third clone (cDNA 6B), 1.5kb in length but lacking a complete 5' end, differed from 6A over a 46 nucleotide stretch which also began at residue 71. Although cDNAs containing this 46 nucleotide splice have been reported to encode for the larger MW form of G(s) α, this protein species was not detectable in canine sarcolemma, a finding possibly related to the fact that cDNA 6B contained a novel sequence 5' to this position as well. Based on the sequences of these three cDNAs as well as S1 nuclease analyses, a minimum of two alternatively spliced exons in addition to duplicated acceptor splice sites need to be involved in the generation of their corresponding mRNA species. Southern blots of genomic DNA were consistent with the existence of a single copy of the G(s) α gene. These findings suggests that the activity of G(s) α is regulated via the expression of multiple isoforms generated via alternative splicing of a single copy gene. Studies are now aimed at characterizing the expression of G(s) α in the hearts of animals with pressure-overload LV hypertrophy and failure.

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