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Essential role for the second extracellular loop in C5a receptor activation.

Jeffery M Klco1, Christina B Wiegand, Kirk Narzinski

  • 1Department of Medicine, Washington University School of Medicine, Campus Box 8127, 660 S. Euclid Avenue, St. Louis, Missouri 63110, USA.

Nature Structural & Molecular Biology
|March 16, 2005
PubMed
Summary

The second extracellular loop (EC2) of G protein-coupled receptors (GPCRs) acts as a negative regulator, stabilizing their inactive state. This study reveals EC2’s crucial role in C5a receptor regulation.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Pharmacology

Background:

  • G protein-coupled receptors (GPCRs) are crucial cell surface receptors involved in numerous physiological processes.
  • A disulfide bridge commonly links the second extracellular loop (EC2) to the third transmembrane helix in over 90% of GPCRs.
  • The precise function of the EC2 region and its associated disulfide bridge in GPCR activation remains incompletely understood.

Purpose of the Study:

  • To investigate the functional significance of the EC2 region and its disulfide bridge in the complement factor 5a receptor (C5aR) activation.
  • To determine if the EC2 region plays a role in regulating C5aR basal activity.

Main Methods:

  • Random saturation mutagenesis was employed on the EC2 region of the C5aR.
  • Functional screening of mutated receptors was performed using a yeast-based system.

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  • Sequence analysis was conducted to identify conserved residues in functional EC2 mutants.

    • Main Results:

    • The cysteine residue forming the disulfide bridge was identified as the sole conserved residue within the EC2 region of functional C5aR mutants.
    • Approximately 80% of the identified functional receptors displayed potent constitutive activity, indicating spontaneous activation.
    • Mutations within the EC2 region led to enhanced receptor activity, suggesting a regulatory role.

    Conclusions:

    • The EC2 region of the C5aR unexpectedly functions as a negative regulator of receptor activation.
    • The disulfide bridge and the EC2 loop likely stabilize the inactive conformation of the C5aR.
    • This finding suggests a potential conserved mechanism where EC2 acts as a negative regulator across various GPCRs.