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Palmitoylation of the human bradykinin B2 receptor influences ligand efficacy

A Pizard1, A Blaukat, S Michineau

  • 1INSERM Unité 367, 17 rue du Fer à Moulin, 75005 Paris, France.

Biochemistry
|December 19, 2001
PubMed

Insights

Palmitoylation of the bradykinin B2 receptor is crucial for its function. Non-palmitoylated receptors show altered responses to antagonists, suggesting palmitoylation stabilizes the receptor-ligand complex in an uncoupled state.

Area of Science:

  • Molecular biology
  • Cellular signaling
  • G protein-coupled receptors

Background:

  • The bradykinin B2 receptor (B2R) is a G protein-coupled receptor involved in various physiological processes.
  • Receptor post-translational modifications, such as palmitoylation, can significantly impact receptor function and signaling.
  • Understanding the role of B2R palmitoylation is essential for elucidating its regulatory mechanisms.

Purpose of the Study:

  • To investigate the role of palmitoylation at cysteine residues 324 and 329 in the carboxyl terminus of the human bradykinin B2 receptor.
  • To determine how these palmitoylation sites affect receptor binding, signaling, and internalization.
  • To explore the functional consequences of a non-palmitoylated B2 receptor on ligand-induced responses.

Main Methods:

  • Site-directed mutagenesis of human B2R to individually or simultaneously mutate cysteines 324 and 329 to glycine.
  • Transfection of mutated receptors into COS-7, CHO-K1, and HEK 293T cells.
  • Assessment of [3H]palmitate incorporation to confirm palmitoylation.
  • Evaluation of [3H]bradykinin binding kinetics, G protein coupling (phospholipase C and A2 activation), and agonist-induced phosphorylation.
  • Measurement of receptor internalization and MAP kinase activation.

Main Results:

  • Wild-type and single cysteine mutants of B2R incorporated [3H]palmitate, while the double mutant did not, confirming palmitoylation at these sites.
  • Palmitoylation did not affect B2R binding kinetics, basal or bradykinin-stimulated phospholipase C and A2 coupling, or agonist-induced phosphorylation.
  • The non-palmitoylated B2 receptor exhibited a 30% reduction in [3H]bradykinin internalization capacity.
  • Antagonists of the wild-type B2R acted as agonists on the non-palmitoylated receptor, triggering phospholipase C and A2 responses.

Conclusions:

  • Palmitoylation of the human bradykinin B2 receptor at cysteines 324 and 329 is not essential for its basal activity, agonist-driven activation, or phosphorylation.
  • Palmitoylation influences B2R internalization, with non-palmitoylated receptors showing reduced capacity.
  • Palmitoylation is critical for maintaining the receptor-ligand complex in an uncoupled conformation, preventing antagonists from eliciting agonist-like responses.

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