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Drug-induced Sensitization of Adenylyl Cyclase: Assay Streamlining and Miniaturization for Small Molecule and siRNA Screening Applications
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Endogenous regulators of G protein signaling differentially modulate full and partial mu-opioid agonists at adenylyl

M J Clark1, J J Linderman, J R Traynor

  • 1Department of Pharmacology, University of Michigan Medical School, Ann Arbor, Michigan, USA.

Molecular Pharmacology
|February 21, 2008
PubMed

Insights

Regulator of G protein signaling (RGS) proteins enhance mu-opioid receptor responses, particularly for low-efficacy agonists. RGS-insensitive mutations increased agonist potency and maximal response, supporting the collision coupling model.

Area of Science:

  • Cellular signaling
  • G protein-coupled receptors
  • Pharmacology

Background:

  • Regulator of G protein signaling (RGS) proteins accelerate GTPase activity of Galpha(i/o) proteins, deactivating signaling.
  • RGS proteins are thought to be more effective in systems with partial agonist responses.
  • The role of RGS proteins in mu-opioid receptor signaling with varying agonist efficacies is not fully understood.

Purpose of the Study:

  • To investigate the role of endogenous RGS proteins in functional responses to mu-opioid agonists with different intrinsic efficacies.
  • To determine how RGS protein interaction affects agonist potency and maximal response.

Main Methods:

  • Stable expression of modified Galpha(i/o) subunits (PTX-sensitive cysteine mutant with or without RGS binding site mutation) in mu-opioid receptor-expressing C6 glioma cells.
  • Treatment with pertussis toxin (PTX) to inactivate endogenous G proteins.
  • Measurement of maximal inhibition of forskolin-stimulated adenylyl cyclase by various mu-opioid agonists.

Main Results:

  • Maximal inhibition by low-efficacy agonists (buprenorphine, nalbuphine) increased in cells with RGS-insensitive Galpha subunits, but not with high-efficacy agonists (DAMGO, morphine).
  • Agonist potency to inhibit adenylyl cyclase increased in cells with RGS-insensitive Galpha subunits, irrespective of agonist efficacy.
  • Data align with the collision coupling model, where RGS-modulated G protein inactivation and agonist efficacy influence steady-state responses.

Conclusions:

  • Endogenous RGS proteins play a significant role in modulating functional responses to mu-opioid receptor agonists, especially those with lower intrinsic efficacy.
  • The interaction between RGS proteins and G proteins is a key determinant of agonist potency and maximal response, as predicted by the collision coupling model.

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