Related Experiment Videos
A limited spectrum of mutations causes constitutive activation of the yeast alpha-factor receptor
C M Sommers1, N P Martin, A Akal-Strader
1Department of Biochemistry and Biophysics, P.O. Box 712, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642, USA.
Abstract:
Activation of G protein coupled receptors (GPCRs) by binding of ligand is the initial event in diverse cellular signaling pathways. To examine the frequency and diversity of mutations that cause constitutive activation of one particular GPCR, the yeast alpha-factor receptor, we screened libraries of random mutations for constitutive alleles. In initial screens for mutant receptor alleles that exhibit signaling in the absence of added ligand, 14 different point mutations were isolated. All of these 14 mutants could be further activated by alpha-factor. Ten of the mutants also acquired the ability to signal in response to binding of desTrp(1)¿Ala(3)ălpha-factor, a peptide that acts as an antagonist toward normal alpha-factor receptors. Of these 10 mutants, at least eight alleles residing in the third, fifth, sixth, and seventh transmembrane segments exhibit bona fide constitutive signaling. The remaining alleles are hypersensitive to alpha-factor rather than constitutive. They can be activated by low concentrations of endogenous alpha-factor present in MATa cells. The strongest constitutively active receptor alleles were recovered multiple times from the mutational libraries, and extensive mutagenesis of certain regions of the alpha-factor receptor did not lead to recovery of any additional constitutive alleles. Thus, only a limited number of mutations is capable of causing constitutive activation of this receptor. Constitutive and hypersensitive signaling by the mutant receptors is partially suppressed by coexpression of normal receptors, consistent with preferential association of the G protein with unactivated receptors.
Insights
Researchers identified a limited number of mutations causing constitutive activation in the yeast alpha-factor receptor, a key G protein-coupled receptor (GPCR). This discovery sheds light on GPCR signaling and potential therapeutic targets.
Area of Science:
- Molecular Biology
- Cellular Signaling
- Biochemistry
Background:
- G protein-coupled receptors (GPCRs) initiate cellular signaling upon ligand binding.
- Understanding mutations causing constitutive GPCR activation is crucial for deciphering signaling pathways.
Purpose of the Study:
- To investigate the frequency and diversity of mutations leading to constitutive activation of the yeast alpha-factor receptor.
- To identify specific mutations responsible for altered receptor activity.
Main Methods:
- Screening of random mutation libraries for constitutive alleles of the yeast alpha-factor receptor.
- Characterization of mutant receptors through activation assays with alpha-factor and antagonist peptides.
- Analysis of mutation locations within transmembrane segments.
Main Results:
- 14 point mutations were isolated, with 10 showing altered responses to alpha-factor and antagonist peptides.
- At least eight mutations in transmembrane segments 3, 5, 6, and 7 conferred bona fide constitutive signaling.
- Other mutations resulted in hypersensitivity to alpha-factor, activated by endogenous ligand.
Conclusions:
- Only a limited set of mutations can cause constitutive activation of the yeast alpha-factor receptor.
- Constitutive and hypersensitive signaling can be partially suppressed by co-expressing normal receptors, suggesting G protein preference for unactivated receptors.