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Updated: Jul 1, 2026

A Kinetic Fluorescence-based Ca2+ Mobilization Assay to Identify G Protein-coupled Receptor Agonists, Antagonists, and Allosteric Modulators
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A Kinetic Fluorescence-based Ca2+ Mobilization Assay to Identify G Protein-coupled Receptor Agonists, Antagonists, and Allosteric Modulators

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The mechanism for ligand activation of the Smoothened G protein-coupled receptor

Ryan D Yu1, Amy-Doan P Vo1, Soo-Kyung Kim1

  • 1Materials and Process Simulation Center, Division of Chemistry & Chemical Engineering, California Institute of Technology, Pasadena, CA 91125.

Proceedings of the National Academy of Sciences of the United States of America
|June 29, 2026
PubMed

Insights

Cholesterol (CHL) binding to Smoothened (SMO) and subsequent G protein interaction initiates the Hedgehog (Hh) signaling pathway. This G protein-first mechanism is crucial for understanding cancer development and targeted therapies.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Cell Signaling

Background:

  • The Smoothened (SMO) receptor is vital in the Hedgehog (Hh) signaling pathway.
  • SMO is an oncoprotein and a key therapeutic target for cancers like basal cell carcinoma.
  • The precise mechanism of SMO activation remains largely unknown.

Purpose of the Study:

  • To elucidate the sequential binding events of G protein and cholesterol (CHL) to SMO.
  • To determine the order of ligand interactions leading to SMO activation.
  • To understand the role of CHL in SMO signaling.

Main Methods:

  • Investigated the sequence of G protein and CHL binding to SMO.
  • Analyzed the role of CHL in SMO activation through CRD and TMD binding.
  • Modeled the G protein-first activation pathway.

Main Results:

  • Results support a G protein-first activation model for SMO in the Hh pathway.
  • CHL initially binds to the cysteine-rich domain (CRD) of inactive SMO.
  • G protein binding to SMO forms a precoupled complex, awaiting Hh ligand interaction with PTCH and increased membrane CHL levels.
  • CHL binding to both CRD and transmembrane domain (TMD) sites promotes G protein opening and downstream signaling.

Conclusions:

  • The study clarifies the activation mechanism of SMO, involving sequential CHL and G protein binding.
  • This G protein-first pathway provides insights into Hh signaling regulation.
  • Understanding this mechanism can inform therapeutic strategies for Hh pathway-driven cancers.
Keywords:
G protein activationcholesterolligandsmoothened GPCR

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