Altered G protein activity in a desensitization-resistant mutant of the Y1 adrenocortical tumor cell line

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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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Drug-induced Sensitization of Adenylyl Cyclase: Assay Streamlining and Miniaturization for Small Molecule and siRNA Screening Applications

Published on: January 27, 2014

C M Colantonio1, W K Kwan, W Czerwinski

  • 1Banting and Best Department of Medical Research, University of Toronto, Ontario, Canada.

Endocrinology
|February 4, 1998
PubMed

Insights

Desensitization resistant (DR) cells prevent receptor uncoupling from G proteins, indicating a G protein defect. This defect alters the activity of the stimulatory G protein (Gs), suggesting a novel factor in guanyl nucleotide exchange.

Area of Science:

  • Cellular signaling pathways
  • G protein-coupled receptor (GPCR) regulation
  • Molecular endocrinology

Background:

  • Agonist-induced desensitization is a key mechanism for regulating cellular responses to external stimuli.
  • Mutant Y1 adrenocortical tumor cells, termed desensitization resistant (DR), exhibit resistance to this desensitization process.
  • The precise molecular basis for DR phenotype, particularly concerning G protein interactions, remains to be fully elucidated.

Purpose of the Study:

  • To investigate whether a defect in G protein function underlies the desensitization resistant (DR) phenotype in Y1 mouse adrenocortical tumor cells.
  • To characterize the specific alterations in G protein activity associated with the DR mutation.
  • To identify the molecular component responsible for the altered G protein activity.

Main Methods:

  • Utilized guanyl-5'-yl imidodiphosphate [Gpp(NH)p] to assess the affinity state of beta2-adrenergic receptors in parent Y1 and DR mutant cells.
  • Performed G protein reconstitution assays using Gs alpha-deficient S49(CYC-) lymphoma cell membranes.
  • Conducted cloning and sequence analysis of Gs alpha transcripts in DR mutant cells.

Main Results:

  • Gpp(NH)p demonstrated a 4-fold greater efficacy in shifting beta2-adrenergic receptors to a low-affinity state in DR mutant cells compared to parent Y1 cells.
  • The effect of Gpp(NH)p on receptor affinity in DR cells was independent of NaCl, unlike in parent Y1 cells.
  • Reconstitution assays confirmed that the DR mutation is associated with altered activity of the stimulatory G protein (Gs).
  • Gs alpha transcripts were found to be normal in DR mutant cells, ruling out mutations in the Gs alpha gene itself.

Conclusions:

  • The desensitization resistant (DR) phenotype is associated with an altered activity of the stimulatory G protein (Gs).
  • The observed defect in G protein activity is not due to mutations in the Gs alpha transcript.
  • These findings suggest that another factor involved in guanyl nucleotide exchange is responsible for the altered G protein activity in DR mutant cells.

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