Co-purification and direct interaction of Ras with caveolin, an integral membrane protein of caveolae microdomains: Detergent-free purification of caveolae membranes

Kenneth S. Song, Shengwen Li, Takashi Okamoto, Lawrence Quilliam, Massimo Sargiacomo, Michael P. Lisanti

Research output: Contribution to journalArticle

889 Citations (Scopus)

Abstract

Caveolae are plasma membrane specializations that have been implicated in signal transduction. Caveolin, a 21-24-kDa integral membrane protein, is a principal structural component of caveolae membranes in vivo. G protein a subunits are concentrated in purified preparations of caveolae membranes, and caveolin interacts directly with multiple G protein α subunits, including Gs, Go, and Gi2. Mutational or pharmacologic activation of G0 subunits prevents the interaction of caveolin with G proteins, indicating that inactive Gα subunits preferentially interact with caveolin. Here, we show that caveolin interacts with another well characterized signal transducer, Ras. Using a detergent-free procedure for purification of caveolin-rich membrane domains and a polyhistidine tagged form of caveolin, we find that Ras and other classes of lipid-modified signaling molecules co-fractionate and co-elute with caveolin. The association of Ras with caveolin was further evaluated using two distinct in vitro binding assays. Wild-type H-Ras interacted with glutathione S-transferase (GST)-caveolin fusion proteins but not with GST alone. Using a battery of GST fusion proteins encoding distinct regions of caveolin, Ras binding activity was localized to a 41-amino acid membrane proximal region of the cytosolic N-terminal domain of caveolin. In addition, reconstituted caveolin-rich membranes (prepared with purified recombinant caveolin and purified lipids) interacted with a soluble form of wild-type H-Ras but failed to interact with mutationally activated soluble H-Ras (G12V). Thus, a single amino acid change (G12V) that constitutively activates Ras prevents or destabilizes this interaction. These results clearly indicate that (i) caveolin is sufficient to recruit soluble Ras onto lipid membranes and (ii) membrane-bound caveolin preferentially interacts with inactive Ras proteins. In direct support of these in vitro studies, we also show that recombinant overexpression of caveolin in intact cells is sufficient to functionally recruit a nonfarnesylated mutant of Ras (C 186S) onto membranes, overcoming the normal requirement for lipid modification of Ras. Taken together, these observations suggest that caveolin may function as a scaffolding protein to localize or sequester certain caveolin-interacting proteins, such as wild-type Ras, within caveolin-rich microdomains of the plasma membrane.

Original languageEnglish
Pages (from-to)9690-9697
Number of pages8
JournalJournal of Biological Chemistry
Volume271
Issue number16
StatePublished - Apr 19 1996

Fingerprint

Caveolins
Caveolae
Detergents
Purification
Membrane Proteins
Membranes
Glutathione Transferase
GTP-Binding Proteins
Protein Subunits
Cell membranes
Lipids
Proteins
Fusion reactions
Cell Membrane

ASJC Scopus subject areas

  • Biochemistry

Cite this

Co-purification and direct interaction of Ras with caveolin, an integral membrane protein of caveolae microdomains : Detergent-free purification of caveolae membranes. / Song, Kenneth S.; Li, Shengwen; Okamoto, Takashi; Quilliam, Lawrence; Sargiacomo, Massimo; Lisanti, Michael P.

In: Journal of Biological Chemistry, Vol. 271, No. 16, 19.04.1996, p. 9690-9697.

Research output: Contribution to journalArticle

@article{9998c03855c14c969fa2d563f3f6165d,
title = "Co-purification and direct interaction of Ras with caveolin, an integral membrane protein of caveolae microdomains: Detergent-free purification of caveolae membranes",
abstract = "Caveolae are plasma membrane specializations that have been implicated in signal transduction. Caveolin, a 21-24-kDa integral membrane protein, is a principal structural component of caveolae membranes in vivo. G protein a subunits are concentrated in purified preparations of caveolae membranes, and caveolin interacts directly with multiple G protein α subunits, including Gs, Go, and Gi2. Mutational or pharmacologic activation of G0 subunits prevents the interaction of caveolin with G proteins, indicating that inactive Gα subunits preferentially interact with caveolin. Here, we show that caveolin interacts with another well characterized signal transducer, Ras. Using a detergent-free procedure for purification of caveolin-rich membrane domains and a polyhistidine tagged form of caveolin, we find that Ras and other classes of lipid-modified signaling molecules co-fractionate and co-elute with caveolin. The association of Ras with caveolin was further evaluated using two distinct in vitro binding assays. Wild-type H-Ras interacted with glutathione S-transferase (GST)-caveolin fusion proteins but not with GST alone. Using a battery of GST fusion proteins encoding distinct regions of caveolin, Ras binding activity was localized to a 41-amino acid membrane proximal region of the cytosolic N-terminal domain of caveolin. In addition, reconstituted caveolin-rich membranes (prepared with purified recombinant caveolin and purified lipids) interacted with a soluble form of wild-type H-Ras but failed to interact with mutationally activated soluble H-Ras (G12V). Thus, a single amino acid change (G12V) that constitutively activates Ras prevents or destabilizes this interaction. These results clearly indicate that (i) caveolin is sufficient to recruit soluble Ras onto lipid membranes and (ii) membrane-bound caveolin preferentially interacts with inactive Ras proteins. In direct support of these in vitro studies, we also show that recombinant overexpression of caveolin in intact cells is sufficient to functionally recruit a nonfarnesylated mutant of Ras (C 186S) onto membranes, overcoming the normal requirement for lipid modification of Ras. Taken together, these observations suggest that caveolin may function as a scaffolding protein to localize or sequester certain caveolin-interacting proteins, such as wild-type Ras, within caveolin-rich microdomains of the plasma membrane.",
author = "Song, {Kenneth S.} and Shengwen Li and Takashi Okamoto and Lawrence Quilliam and Massimo Sargiacomo and Lisanti, {Michael P.}",
year = "1996",
month = "4",
day = "19",
language = "English",
volume = "271",
pages = "9690--9697",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "16",

}

TY - JOUR

T1 - Co-purification and direct interaction of Ras with caveolin, an integral membrane protein of caveolae microdomains

T2 - Detergent-free purification of caveolae membranes

AU - Song, Kenneth S.

AU - Li, Shengwen

AU - Okamoto, Takashi

AU - Quilliam, Lawrence

AU - Sargiacomo, Massimo

AU - Lisanti, Michael P.

PY - 1996/4/19

Y1 - 1996/4/19

N2 - Caveolae are plasma membrane specializations that have been implicated in signal transduction. Caveolin, a 21-24-kDa integral membrane protein, is a principal structural component of caveolae membranes in vivo. G protein a subunits are concentrated in purified preparations of caveolae membranes, and caveolin interacts directly with multiple G protein α subunits, including Gs, Go, and Gi2. Mutational or pharmacologic activation of G0 subunits prevents the interaction of caveolin with G proteins, indicating that inactive Gα subunits preferentially interact with caveolin. Here, we show that caveolin interacts with another well characterized signal transducer, Ras. Using a detergent-free procedure for purification of caveolin-rich membrane domains and a polyhistidine tagged form of caveolin, we find that Ras and other classes of lipid-modified signaling molecules co-fractionate and co-elute with caveolin. The association of Ras with caveolin was further evaluated using two distinct in vitro binding assays. Wild-type H-Ras interacted with glutathione S-transferase (GST)-caveolin fusion proteins but not with GST alone. Using a battery of GST fusion proteins encoding distinct regions of caveolin, Ras binding activity was localized to a 41-amino acid membrane proximal region of the cytosolic N-terminal domain of caveolin. In addition, reconstituted caveolin-rich membranes (prepared with purified recombinant caveolin and purified lipids) interacted with a soluble form of wild-type H-Ras but failed to interact with mutationally activated soluble H-Ras (G12V). Thus, a single amino acid change (G12V) that constitutively activates Ras prevents or destabilizes this interaction. These results clearly indicate that (i) caveolin is sufficient to recruit soluble Ras onto lipid membranes and (ii) membrane-bound caveolin preferentially interacts with inactive Ras proteins. In direct support of these in vitro studies, we also show that recombinant overexpression of caveolin in intact cells is sufficient to functionally recruit a nonfarnesylated mutant of Ras (C 186S) onto membranes, overcoming the normal requirement for lipid modification of Ras. Taken together, these observations suggest that caveolin may function as a scaffolding protein to localize or sequester certain caveolin-interacting proteins, such as wild-type Ras, within caveolin-rich microdomains of the plasma membrane.

AB - Caveolae are plasma membrane specializations that have been implicated in signal transduction. Caveolin, a 21-24-kDa integral membrane protein, is a principal structural component of caveolae membranes in vivo. G protein a subunits are concentrated in purified preparations of caveolae membranes, and caveolin interacts directly with multiple G protein α subunits, including Gs, Go, and Gi2. Mutational or pharmacologic activation of G0 subunits prevents the interaction of caveolin with G proteins, indicating that inactive Gα subunits preferentially interact with caveolin. Here, we show that caveolin interacts with another well characterized signal transducer, Ras. Using a detergent-free procedure for purification of caveolin-rich membrane domains and a polyhistidine tagged form of caveolin, we find that Ras and other classes of lipid-modified signaling molecules co-fractionate and co-elute with caveolin. The association of Ras with caveolin was further evaluated using two distinct in vitro binding assays. Wild-type H-Ras interacted with glutathione S-transferase (GST)-caveolin fusion proteins but not with GST alone. Using a battery of GST fusion proteins encoding distinct regions of caveolin, Ras binding activity was localized to a 41-amino acid membrane proximal region of the cytosolic N-terminal domain of caveolin. In addition, reconstituted caveolin-rich membranes (prepared with purified recombinant caveolin and purified lipids) interacted with a soluble form of wild-type H-Ras but failed to interact with mutationally activated soluble H-Ras (G12V). Thus, a single amino acid change (G12V) that constitutively activates Ras prevents or destabilizes this interaction. These results clearly indicate that (i) caveolin is sufficient to recruit soluble Ras onto lipid membranes and (ii) membrane-bound caveolin preferentially interacts with inactive Ras proteins. In direct support of these in vitro studies, we also show that recombinant overexpression of caveolin in intact cells is sufficient to functionally recruit a nonfarnesylated mutant of Ras (C 186S) onto membranes, overcoming the normal requirement for lipid modification of Ras. Taken together, these observations suggest that caveolin may function as a scaffolding protein to localize or sequester certain caveolin-interacting proteins, such as wild-type Ras, within caveolin-rich microdomains of the plasma membrane.

UR - http://www.scopus.com/inward/record.url?scp=0029912981&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0029912981&partnerID=8YFLogxK

M3 - Article

C2 - 8621645

AN - SCOPUS:0029912981

VL - 271

SP - 9690

EP - 9697

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 16

ER -