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Adenylyl cyclase inhibition and altered G protein subunit expression and ADP-ribosylation patterns in tissues and
U Rudolph1, K Spicher, L Birnbaumer
1Department of Cell Biology, Baylor College of Medicine, Houston, TX 77030, USA.
Abstract:
The inhibition of alpha i2-/- mouse cardiac isoproterenol-stimulated adenylyl cyclase (AC; EC 4.6.1.1) activity by carbachol and that of alpha i2-/- adipocyte AC by phenylisopropyladenosine (PIA), prostaglandin E2, and nicotinic acid were partially, but not completely, inhibited. While the inhibition of cardiac AC was affected in all alpha i2-/- animals tested, only 50% of the alpha i2-/- animals showed an impaired inhibition of adipocyte AC, indicative of a partial penetrance of this phenotype. In agreement with previous results, the data show that Gi2 mediates hormonal inhibition of AC and that Gi3 and/or Gi1 is capable of doing the same but with a lower efficacy. Disruption of the alpha i2 gene affected about equally the actions of all the receptors studied, indicating that none of them exhibits a striking specificity for one type of Gi over another and that receptors are likely to he selective rather than specific in their interaction with functionally homologous G proteins (e.g., Gi1, Gi2, Gi3). Western analysis of G protein subunit levels in simian virus 40-transformed primary embryonic fibroblasts from alpha i2+/+ and alpha i2-/- animals showed that alpha i2 accounts for about 50% of the immunopositive G protein alpha subunits and that loss of the alpha i2 is accompanied by a parallel reduction in G beta 35 and G beta 36 subunits and by a 30-50% increase in alpha i3. This suggests that G beta-gamma levels may be regulated passively through differential rates of turnover in their free vs. trimeric states. The existence of compensatory increase(s) in alpha i subunit expression raises the possibility that the lack of effect of a missing alpha i2 on AC inhibition in adipocytes of some alpha i2-/- animals may be the reflection of a more pronounced compensatory expression of alpha i3 and/or alpha i1.
Insights
The study reveals that while Gi2 protein primarily inhibits adenylyl cyclase (AC), other Gi proteins can compensate, especially when alpha i2 is absent. This compensation, however, is incomplete and varies between cell types.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Signaling
Background:
- G proteins, specifically the Gi family, are crucial regulators of adenylyl cyclase (AC) activity.
- Gi2 is known to mediate hormonal inhibition of AC, but the roles of Gi1 and Gi3 are less understood.
- Understanding the specificity and redundancy of Gi proteins in AC inhibition is important for deciphering cellular signaling pathways.
Purpose of the Study:
- To investigate the role of the alpha i2 gene in mediating hormonal inhibition of adenylyl cyclase (AC).
- To determine the compensatory roles of other Gi proteins (Gi1, Gi3) in the absence of functional alpha i2.
- To analyze the impact of alpha i2 gene disruption on the expression of other G protein subunits.
Main Methods:
- Utilized alpha i2-/- knockout mice to study cardiac and adipocyte adenylyl cyclase (AC) activity.
- Assessed AC inhibition by various agonists (carbachol, PIA, prostaglandin E2, nicotinic acid).
- Performed Western blot analysis to quantify G protein alpha and beta subunit levels in fibroblasts from alpha i2+/+ and alpha i2-/- animals.
Main Results:
- Inhibition of cardiac AC was impaired in all alpha i2-/- mice, while adipocyte AC inhibition was only partially affected in 50% of animals.
- Gi2 mediates AC inhibition, with Gi3 and/or Gi1 showing lower efficacy.
- Loss of alpha i2 led to reduced G beta subunits and a compensatory increase in alpha i3 expression, suggesting passive regulation of G beta-gamma levels.
Conclusions:
- Gi2 is a primary mediator of AC inhibition, but Gi1 and Gi3 can compensate, albeit with lower efficacy.
- The partial penetrance of the adipocyte AC inhibition phenotype in alpha i2-/- mice is likely due to compensatory expression of other Gi alpha subunits.
- Receptor interactions with Gi proteins are selective rather than strictly specific, allowing for functional redundancy.