Related Experiment Videos
Altered G protein activity in a desensitization-resistant mutant of the Y1 adrenocortical tumor cell line
C M Colantonio1, W K Kwan, W Czerwinski
1Banting and Best Department of Medical Research, University of Toronto, Ontario, Canada.
Abstract:
Mutant isolates [designated desensitization resistant (DR)] from the Y1 mouse adrenocortical tumor cell line resist agonist-induced desensitization of adenylyl cyclase by preventing the uncoupling of receptors from their guanyl nucleotide-binding regulatory G proteins. In this study, we tested the hypothesis that an underlying G protein defect is associated with the DR phenotype. We found that the G protein reagent guanyl-5'-yl imidodiphosphate [Gpp(NH)p] shifted beta2-adrenergic receptors from a high affinity state to a low affinity state 4-fold more effectively in mutant DR cells than in parent Y1 cells. In the DR mutant, Gpp(NH)p was able to shift receptors to a low affinity state in the absence of NaCl, whereas the effect of Gpp(NH)p in parent Y1 cells was dependent upon the presence of NaCl. Moreover, these differences in sensitivity to Gpp(NH)p and NaCl were transferred to Gs alpha-deficient S49(CYC-) lymphoma cell membranes in G protein reconstitution assays. These observations suggested that the DR mutation was associated with altered activity of the stimulatory G protein, Gs. Cloning and sequence analysis demonstrated that Gs alpha transcripts in the DR mutant were normal, suggesting that another factor involved in guanyl nucleotide exchange is responsible for the altered G protein activity in DR mutant cells.
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.