Caveolin-1 interacts directly with dynamin-2

Qing Yao, Jing Chen, Hong Cao, James D. Orth, J. Michael McCaffery, Radu Virgil Stan, Mark A Mc Niven

Research output: Contribution to journalArticle

75 Citations (Scopus)

Abstract

Caveolin is the principal component of caveolae in vivo. In addition to a structural role, it is believed to play a scaffolding function to organize and inactivate signaling molecules that are concentrated on the cytoplasmic surface of caveolar membranes. The large GTPase dynamin has been shown to mediate the scission of caveolae from the plasma membrane, although it is unclear if dynamin interacts directly with caveolin or via accessory proteins. Therefore, the goal of this study was to test whether dynamin associates with caveolae via a direct binding to the caveolin 1 (Cav1) protein. Immunoelectron microscopy of lung endothelium or a cultured hepatocyte cell line stained with antibodies for Dyn2 and Cav1 shows that these proteins co-localize to caveolae. To further define this interaction biochemically, in vitro experiments were performed using glutathione-S-transferase (GST)-Dyn2 and GST-Cav1 fusion proteins, which demonstrated a direct interaction between these proteins. This interaction appears to be mediated by the proline-arginine-rich domain (PRD) of Dyn2, as a GST-PRD fragment binds Cav1 while GST-Dyn2ΔPRD does not. Further, in vitro binding studies using two Dyn2 spliced forms and Cav1 peptides immobilized on paper identify specific domains of Cav1 that bind Dyn2. Interestingly, these Cav1-binding domains differ markedly between two spliced variant forms of Dyn2. In support of these distinctive physical interactions, we find that the different Dyn2 forms, when expressed as GTPase-defective mutants, exert markedly different inhibitory effects on caveolae internalization, as assayed by cholera toxin uptake. These studies provide the first evidence for a direct interaction between dynamin and the caveolin coat, and demonstrate a selectivity of one Dyn2 form toward the caveolae-mediated endocytosis.

Original languageEnglish (US)
Pages (from-to)491-501
Number of pages11
JournalJournal of Molecular Biology
Volume348
Issue number2
DOIs
StatePublished - Apr 29 2005

Fingerprint

Dynamin II
Caveolin 1
Caveolae
Caveolins
Dynamins
Glutathione Transferase
GTP Phosphohydrolases
Proline
Arginine
Proteins
Immunoelectron Microscopy
Cholera Toxin
Endocytosis
Endothelium
Hepatocytes
Cultured Cells
Cell Membrane
Cell Line
Lung
Peptides

Keywords

  • Caveolae
  • Caveolin-dynamin interactions
  • Dynamin
  • Protein-protein binding
  • Vesicle formation

ASJC Scopus subject areas

  • Virology

Cite this

Yao, Q., Chen, J., Cao, H., Orth, J. D., McCaffery, J. M., Stan, R. V., & Mc Niven, M. A. (2005). Caveolin-1 interacts directly with dynamin-2. Journal of Molecular Biology, 348(2), 491-501. https://doi.org/10.1016/j.jmb.2005.02.003

Caveolin-1 interacts directly with dynamin-2. / Yao, Qing; Chen, Jing; Cao, Hong; Orth, James D.; McCaffery, J. Michael; Stan, Radu Virgil; Mc Niven, Mark A.

In: Journal of Molecular Biology, Vol. 348, No. 2, 29.04.2005, p. 491-501.

Research output: Contribution to journalArticle

Yao, Q, Chen, J, Cao, H, Orth, JD, McCaffery, JM, Stan, RV & Mc Niven, MA 2005, 'Caveolin-1 interacts directly with dynamin-2', Journal of Molecular Biology, vol. 348, no. 2, pp. 491-501. https://doi.org/10.1016/j.jmb.2005.02.003
Yao Q, Chen J, Cao H, Orth JD, McCaffery JM, Stan RV et al. Caveolin-1 interacts directly with dynamin-2. Journal of Molecular Biology. 2005 Apr 29;348(2):491-501. https://doi.org/10.1016/j.jmb.2005.02.003
Yao, Qing ; Chen, Jing ; Cao, Hong ; Orth, James D. ; McCaffery, J. Michael ; Stan, Radu Virgil ; Mc Niven, Mark A. / Caveolin-1 interacts directly with dynamin-2. In: Journal of Molecular Biology. 2005 ; Vol. 348, No. 2. pp. 491-501.
@article{3e89400aa7e845e5a9b8d39364c971da,
title = "Caveolin-1 interacts directly with dynamin-2",
abstract = "Caveolin is the principal component of caveolae in vivo. In addition to a structural role, it is believed to play a scaffolding function to organize and inactivate signaling molecules that are concentrated on the cytoplasmic surface of caveolar membranes. The large GTPase dynamin has been shown to mediate the scission of caveolae from the plasma membrane, although it is unclear if dynamin interacts directly with caveolin or via accessory proteins. Therefore, the goal of this study was to test whether dynamin associates with caveolae via a direct binding to the caveolin 1 (Cav1) protein. Immunoelectron microscopy of lung endothelium or a cultured hepatocyte cell line stained with antibodies for Dyn2 and Cav1 shows that these proteins co-localize to caveolae. To further define this interaction biochemically, in vitro experiments were performed using glutathione-S-transferase (GST)-Dyn2 and GST-Cav1 fusion proteins, which demonstrated a direct interaction between these proteins. This interaction appears to be mediated by the proline-arginine-rich domain (PRD) of Dyn2, as a GST-PRD fragment binds Cav1 while GST-Dyn2ΔPRD does not. Further, in vitro binding studies using two Dyn2 spliced forms and Cav1 peptides immobilized on paper identify specific domains of Cav1 that bind Dyn2. Interestingly, these Cav1-binding domains differ markedly between two spliced variant forms of Dyn2. In support of these distinctive physical interactions, we find that the different Dyn2 forms, when expressed as GTPase-defective mutants, exert markedly different inhibitory effects on caveolae internalization, as assayed by cholera toxin uptake. These studies provide the first evidence for a direct interaction between dynamin and the caveolin coat, and demonstrate a selectivity of one Dyn2 form toward the caveolae-mediated endocytosis.",
keywords = "Caveolae, Caveolin-dynamin interactions, Dynamin, Protein-protein binding, Vesicle formation",
author = "Qing Yao and Jing Chen and Hong Cao and Orth, {James D.} and McCaffery, {J. Michael} and Stan, {Radu Virgil} and {Mc Niven}, {Mark A}",
year = "2005",
month = "4",
day = "29",
doi = "10.1016/j.jmb.2005.02.003",
language = "English (US)",
volume = "348",
pages = "491--501",
journal = "Journal of Molecular Biology",
issn = "0022-2836",
publisher = "Academic Press Inc.",
number = "2",

}

TY - JOUR

T1 - Caveolin-1 interacts directly with dynamin-2

AU - Yao, Qing

AU - Chen, Jing

AU - Cao, Hong

AU - Orth, James D.

AU - McCaffery, J. Michael

AU - Stan, Radu Virgil

AU - Mc Niven, Mark A

PY - 2005/4/29

Y1 - 2005/4/29

N2 - Caveolin is the principal component of caveolae in vivo. In addition to a structural role, it is believed to play a scaffolding function to organize and inactivate signaling molecules that are concentrated on the cytoplasmic surface of caveolar membranes. The large GTPase dynamin has been shown to mediate the scission of caveolae from the plasma membrane, although it is unclear if dynamin interacts directly with caveolin or via accessory proteins. Therefore, the goal of this study was to test whether dynamin associates with caveolae via a direct binding to the caveolin 1 (Cav1) protein. Immunoelectron microscopy of lung endothelium or a cultured hepatocyte cell line stained with antibodies for Dyn2 and Cav1 shows that these proteins co-localize to caveolae. To further define this interaction biochemically, in vitro experiments were performed using glutathione-S-transferase (GST)-Dyn2 and GST-Cav1 fusion proteins, which demonstrated a direct interaction between these proteins. This interaction appears to be mediated by the proline-arginine-rich domain (PRD) of Dyn2, as a GST-PRD fragment binds Cav1 while GST-Dyn2ΔPRD does not. Further, in vitro binding studies using two Dyn2 spliced forms and Cav1 peptides immobilized on paper identify specific domains of Cav1 that bind Dyn2. Interestingly, these Cav1-binding domains differ markedly between two spliced variant forms of Dyn2. In support of these distinctive physical interactions, we find that the different Dyn2 forms, when expressed as GTPase-defective mutants, exert markedly different inhibitory effects on caveolae internalization, as assayed by cholera toxin uptake. These studies provide the first evidence for a direct interaction between dynamin and the caveolin coat, and demonstrate a selectivity of one Dyn2 form toward the caveolae-mediated endocytosis.

AB - Caveolin is the principal component of caveolae in vivo. In addition to a structural role, it is believed to play a scaffolding function to organize and inactivate signaling molecules that are concentrated on the cytoplasmic surface of caveolar membranes. The large GTPase dynamin has been shown to mediate the scission of caveolae from the plasma membrane, although it is unclear if dynamin interacts directly with caveolin or via accessory proteins. Therefore, the goal of this study was to test whether dynamin associates with caveolae via a direct binding to the caveolin 1 (Cav1) protein. Immunoelectron microscopy of lung endothelium or a cultured hepatocyte cell line stained with antibodies for Dyn2 and Cav1 shows that these proteins co-localize to caveolae. To further define this interaction biochemically, in vitro experiments were performed using glutathione-S-transferase (GST)-Dyn2 and GST-Cav1 fusion proteins, which demonstrated a direct interaction between these proteins. This interaction appears to be mediated by the proline-arginine-rich domain (PRD) of Dyn2, as a GST-PRD fragment binds Cav1 while GST-Dyn2ΔPRD does not. Further, in vitro binding studies using two Dyn2 spliced forms and Cav1 peptides immobilized on paper identify specific domains of Cav1 that bind Dyn2. Interestingly, these Cav1-binding domains differ markedly between two spliced variant forms of Dyn2. In support of these distinctive physical interactions, we find that the different Dyn2 forms, when expressed as GTPase-defective mutants, exert markedly different inhibitory effects on caveolae internalization, as assayed by cholera toxin uptake. These studies provide the first evidence for a direct interaction between dynamin and the caveolin coat, and demonstrate a selectivity of one Dyn2 form toward the caveolae-mediated endocytosis.

KW - Caveolae

KW - Caveolin-dynamin interactions

KW - Dynamin

KW - Protein-protein binding

KW - Vesicle formation

UR - http://www.scopus.com/inward/record.url?scp=16244399830&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=16244399830&partnerID=8YFLogxK

U2 - 10.1016/j.jmb.2005.02.003

DO - 10.1016/j.jmb.2005.02.003

M3 - Article

VL - 348

SP - 491

EP - 501

JO - Journal of Molecular Biology

JF - Journal of Molecular Biology

SN - 0022-2836

IS - 2

ER -