Amino- and carboxy-terminal deletion mutants of gsα are localized to the particulate fraction of transfected COS cells

Yong Sung Juhnn, Teresa L Z Jones, Allen M. Spiegel

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

To elucidate the structural basis for membrane attachment of the α subunit of the stimulatory G protein (Gsα), mutant Gsα cDNAs with deletions of amino acid residues in the amino and/or carboxy termini were transiently expressed in COS-7 cells. The particulate and soluble fractions prepared from these cells were analyzed by immunoblot using peptide specific antibodies to monitor distribution of the expressed proteins. Transfection of mutant forms of Gsa with either 26 amino terminal residues deleted (Δ3-28) or with 59 amino terminal residues deleted (Δ1-59) resulted in immunoreactive proteins which localized primarily to the particulate fraction. Similarly, mutants with 10 (Δ385-394), 32 (Δ353-384), or 42 (Δ353-394) amino acid residues deleted from the carboxy terminus also localized to the particulate fraction, as did a mutant form of Gsα lacking amino acid residues at both the amino and carboxy termini (Δ3-28)/(Δ353-384). Mutant and wild type forms of Gsα demonstrated a similar degree of tightness in their binding to membranes as demonstrated by treatment with 2.5 M NaCl or 6 M urea, but some mutant forms were relatively resistant compared with wild type Gsα to solubilization by 15 mM NaOH or 1% sodium cholate. We conclude that: (a) deletion of significant portions of the amino and/or carboxyl terminus of Gsα is still compatible with protein expression; (b) deletion of these regions is insufficient to cause cytosolic localization of the expressed protein. The basis of Gsα membrane targeting remains to be elucidated.

Original languageEnglish (US)
Pages (from-to)523-530
Number of pages8
JournalJournal of Cell Biology
Volume119
Issue number3
StatePublished - Nov 1992
Externally publishedYes

Fingerprint

COS Cells
Amino Acids
Membranes
Proteins
Gs GTP-Binding Protein alpha Subunits
Sodium Cholate
Transfection
Urea
Complementary DNA
Peptides
Antibodies

ASJC Scopus subject areas

  • Cell Biology

Cite this

Amino- and carboxy-terminal deletion mutants of gsα are localized to the particulate fraction of transfected COS cells. / Juhnn, Yong Sung; Jones, Teresa L Z; Spiegel, Allen M.

In: Journal of Cell Biology, Vol. 119, No. 3, 11.1992, p. 523-530.

Research output: Contribution to journalArticle

@article{7f3506cc89bd40659d09dd667933032f,
title = "Amino- and carboxy-terminal deletion mutants of gsα are localized to the particulate fraction of transfected COS cells",
abstract = "To elucidate the structural basis for membrane attachment of the α subunit of the stimulatory G protein (Gsα), mutant Gsα cDNAs with deletions of amino acid residues in the amino and/or carboxy termini were transiently expressed in COS-7 cells. The particulate and soluble fractions prepared from these cells were analyzed by immunoblot using peptide specific antibodies to monitor distribution of the expressed proteins. Transfection of mutant forms of Gsa with either 26 amino terminal residues deleted (Δ3-28) or with 59 amino terminal residues deleted (Δ1-59) resulted in immunoreactive proteins which localized primarily to the particulate fraction. Similarly, mutants with 10 (Δ385-394), 32 (Δ353-384), or 42 (Δ353-394) amino acid residues deleted from the carboxy terminus also localized to the particulate fraction, as did a mutant form of Gsα lacking amino acid residues at both the amino and carboxy termini (Δ3-28)/(Δ353-384). Mutant and wild type forms of Gsα demonstrated a similar degree of tightness in their binding to membranes as demonstrated by treatment with 2.5 M NaCl or 6 M urea, but some mutant forms were relatively resistant compared with wild type Gsα to solubilization by 15 mM NaOH or 1{\%} sodium cholate. We conclude that: (a) deletion of significant portions of the amino and/or carboxyl terminus of Gsα is still compatible with protein expression; (b) deletion of these regions is insufficient to cause cytosolic localization of the expressed protein. The basis of Gsα membrane targeting remains to be elucidated.",
author = "Juhnn, {Yong Sung} and Jones, {Teresa L Z} and Spiegel, {Allen M.}",
year = "1992",
month = "11",
language = "English (US)",
volume = "119",
pages = "523--530",
journal = "Journal of Cell Biology",
issn = "0021-9525",
publisher = "Rockefeller University Press",
number = "3",

}

TY - JOUR

T1 - Amino- and carboxy-terminal deletion mutants of gsα are localized to the particulate fraction of transfected COS cells

AU - Juhnn, Yong Sung

AU - Jones, Teresa L Z

AU - Spiegel, Allen M.

PY - 1992/11

Y1 - 1992/11

N2 - To elucidate the structural basis for membrane attachment of the α subunit of the stimulatory G protein (Gsα), mutant Gsα cDNAs with deletions of amino acid residues in the amino and/or carboxy termini were transiently expressed in COS-7 cells. The particulate and soluble fractions prepared from these cells were analyzed by immunoblot using peptide specific antibodies to monitor distribution of the expressed proteins. Transfection of mutant forms of Gsa with either 26 amino terminal residues deleted (Δ3-28) or with 59 amino terminal residues deleted (Δ1-59) resulted in immunoreactive proteins which localized primarily to the particulate fraction. Similarly, mutants with 10 (Δ385-394), 32 (Δ353-384), or 42 (Δ353-394) amino acid residues deleted from the carboxy terminus also localized to the particulate fraction, as did a mutant form of Gsα lacking amino acid residues at both the amino and carboxy termini (Δ3-28)/(Δ353-384). Mutant and wild type forms of Gsα demonstrated a similar degree of tightness in their binding to membranes as demonstrated by treatment with 2.5 M NaCl or 6 M urea, but some mutant forms were relatively resistant compared with wild type Gsα to solubilization by 15 mM NaOH or 1% sodium cholate. We conclude that: (a) deletion of significant portions of the amino and/or carboxyl terminus of Gsα is still compatible with protein expression; (b) deletion of these regions is insufficient to cause cytosolic localization of the expressed protein. The basis of Gsα membrane targeting remains to be elucidated.

AB - To elucidate the structural basis for membrane attachment of the α subunit of the stimulatory G protein (Gsα), mutant Gsα cDNAs with deletions of amino acid residues in the amino and/or carboxy termini were transiently expressed in COS-7 cells. The particulate and soluble fractions prepared from these cells were analyzed by immunoblot using peptide specific antibodies to monitor distribution of the expressed proteins. Transfection of mutant forms of Gsa with either 26 amino terminal residues deleted (Δ3-28) or with 59 amino terminal residues deleted (Δ1-59) resulted in immunoreactive proteins which localized primarily to the particulate fraction. Similarly, mutants with 10 (Δ385-394), 32 (Δ353-384), or 42 (Δ353-394) amino acid residues deleted from the carboxy terminus also localized to the particulate fraction, as did a mutant form of Gsα lacking amino acid residues at both the amino and carboxy termini (Δ3-28)/(Δ353-384). Mutant and wild type forms of Gsα demonstrated a similar degree of tightness in their binding to membranes as demonstrated by treatment with 2.5 M NaCl or 6 M urea, but some mutant forms were relatively resistant compared with wild type Gsα to solubilization by 15 mM NaOH or 1% sodium cholate. We conclude that: (a) deletion of significant portions of the amino and/or carboxyl terminus of Gsα is still compatible with protein expression; (b) deletion of these regions is insufficient to cause cytosolic localization of the expressed protein. The basis of Gsα membrane targeting remains to be elucidated.

UR - http://www.scopus.com/inward/record.url?scp=0026764981&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0026764981&partnerID=8YFLogxK

M3 - Article

C2 - 1400589

AN - SCOPUS:0026764981

VL - 119

SP - 523

EP - 530

JO - Journal of Cell Biology

JF - Journal of Cell Biology

SN - 0021-9525

IS - 3

ER -