Dynamic regulation of a GPCR-tetraspanin-G protein complex on intact cells: central role of CD81 in facilitating GPR56-Galpha q/11 association

Mol Biol Cell. 2004 May;15(5):2375-87. doi: 10.1091/mbc.e03-12-0886. Epub 2004 Mar 5.

Abstract

By means of a variety of intracellular scaffolding proteins, a vast number of heterotrimeric G protein-coupled receptors (GPCRs) may achieve specificity in signaling through a much smaller number of heterotrimeric G proteins. Members of the tetraspanin family organize extensive complexes of cell surface proteins and thus have the potential to act as GPCR scaffolds; however, tetraspanin-GPCR complexes had not previously been described. We now show that a GPCR, GPR56/TM7XN1, and heterotrimeric G protein subunits, Galpha(q), Galpha(11), and Gbeta, associate specifically with tetraspanins and CD81, but not with other tetraspanins. CD9 Complexes of GPR56 with CD9 and CD81 remained intact when fully solubilized and were resistant to cholesterol depletion. Hence they do not depend on detergent-insoluble, raft-like membrane microdomains for stability. A central role for CD81 in promoting or stabilizing a GPR56-CD81-Galpha(q/11) complex was revealed by CD81 immunodepletion and reexpression experiments. Finally, antibody engagement of cell surface CD81 or cell activation with phorbol ester revealed two distinct mechanisms by which GPR56-CD81-Galpha(q/11) complexes can be dynamically regulated. These data reveal a potential role for tetraspanins CD9 and CD81 as GPCR scaffolding proteins.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Antibodies / immunology
  • Antigens, CD / physiology*
  • Cell Line
  • GTP-Binding Protein alpha Subunits, Gq-G11 / analysis
  • GTP-Binding Protein alpha Subunits, Gq-G11 / metabolism*
  • GTP-Binding Protein beta Subunits / analysis
  • GTP-Binding Protein beta Subunits / metabolism
  • Humans
  • Immunoglobulins / analysis
  • Immunoglobulins / metabolism
  • Immunoprecipitation
  • Membrane Proteins
  • Neoplasm Proteins / analysis
  • Neoplasm Proteins / metabolism
  • Phorbol Esters / metabolism
  • Phorbol Esters / pharmacology
  • Protein Binding
  • Protein Interaction Mapping
  • Receptors, G-Protein-Coupled / analysis
  • Receptors, G-Protein-Coupled / metabolism*
  • Receptors, G-Protein-Coupled / physiology
  • Signal Transduction
  • Tetraspanin 28

Substances

  • ADGRG1 protein, human
  • Antibodies
  • Antigens, CD
  • CD81 protein, human
  • Cd81 protein, rat
  • GTP-Binding Protein beta Subunits
  • IGSF8 protein, human
  • Immunoglobulins
  • Membrane Proteins
  • Neoplasm Proteins
  • Phorbol Esters
  • Ptgfrn protein, rat
  • Receptors, G-Protein-Coupled
  • Tetraspanin 28
  • GTP-Binding Protein alpha Subunits, Gq-G11