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Characterization of G Protein-coupled Receptors by a Fluorescence-based Calcium Mobilization Assay
11:49

Characterization of G Protein-coupled Receptors by a Fluorescence-based Calcium Mobilization Assay

Published on: July 28, 2014

Receptor-mediated activation of Gsalpha: evidence for intramolecular signal transduction

S R Marsh1, G Grishina, P T Wilson

  • 1Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06520-8026, USA.

Molecular Pharmacology
|June 17, 1998
PubMed

Insights

Investigating G protein activation, this study used mutagenesis to find key residues in the alpha subunit. These findings reveal how cell surface receptors trigger signaling pathways by influencing G protein function.

Area of Science:

  • Molecular Biology
  • Cell Signaling
  • Biochemistry

Background:

  • Cell surface receptors initiate intracellular signaling cascades by activating heterotrimeric G proteins.
  • The alpha subunit of G proteins plays a critical role in mediating this activation process.

Purpose of the Study:

  • To elucidate the mechanism of receptor-mediated activation of G proteins.
  • To identify specific residues within the alpha subunit's carboxyl-terminal domain involved in this activation.

Main Methods:

  • Scanning mutagenesis of the carboxyl-terminal 40% of the alpha subunit (residues 236-394).
  • Assay of receptor-dependent cAMP synthesis in cyc- S49 lymphoma cells lacking endogenous alpha subunits.
  • Construction and testing of alpha subunit chimeras (alphas/alphai2/alphas).

Main Results:

  • Four critical regions within the alpha subunit were identified where mutations impaired receptor-mediated activation.
  • Carboxyl-terminal residues are implicated as potential receptor contact sites.
  • Residues involved in GDP binding and signal relay across domain interfaces were identified, including switch III.
  • A chimera study demonstrated the importance of domain interface interactions for activation.

Conclusions:

  • Specific residues and their spatial arrangement within the alpha subunit are crucial for translating receptor binding into G protein activation.
  • Complementary interactions across the GTPase and helical domain interface facilitate GDP release.
  • This research provides mechanistic insights into G protein signaling initiation.

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