Mutations on the Switch III region and the alpha3 helix of Galpha&lt;sub&gt;16&lt;/sub&gt; differentially affect receptor coupling and regulation of downstream effectors

May Y M Yu,Yung H Wong,Maurice K C Ho,Andrew M F Liu

doi:10.1186/1750-2187-3-17

Abstract

BackgroundGα16 can activate phospholipase Cβ (PLCβ) directly like Gαq. It also couples to tetratricopeptide repeat 1 (TPR1) which is linked to Ras activation. It is unknown whether PLCβ and TPR1 interact with the same regions on Gα16. Previous studies on Gαq have defined two minimal clusters of amino acids that are essential for the coupling to PLCβ. Cognate residues in Gα16 might also be essential for interacting with PLCβ, and possibly contribute to TPR1 interaction and other signaling events.ResultsAlanine mutations were introduced to the two amino acid clusters (246–248 and 259–260) in the switch III region and α3 helix of Gα16. Regulations of PLCβ and STAT3 were partially weakened by each cluster mutant. A mutant harboring mutations at both clusters generally produced stronger suppressions. Activation of Jun N-terminal kinase (JNK) by Gα16 was completely abolished by mutating either clusters. Contrastingly, phosphorylations of extracellular signal-regulated kinase (ERK) and nuclear factor κB (NF-κB) were not significantly affected by these mutations. The interactions between the mutants and PLCβ2 and TPR1 were also reduced in co-immunoprecipitation assays. Coupling between G16 and different categories of receptors was impaired by the mutations, with the effect of switch III mutations being more pronounced than those in the α3 helix. Mutations of both clusters almost completely abolished the receptor coupling and prevent receptor-induced Gβγ release.ConclusionThe integrity of the switch III region and α3 helix of Gα16 is critical for the activation of PLCβ, STAT3, and JNK but not ERK or NF-κB. Binding of Gα16 to PLCβ2 or TPR1 was reduced by the mutations of either cluster. The same region could also differentially affect the effectiveness of receptor coupling to G16. The studied region was shown to bear multiple functionally important roles of G16.

Highlights

Gα16 can activate phospholipase Cβ (PLCβ) directly like Gαq
The results suggested that the two regions of interest were important for the activation of PLCβ, STAT3, Jun N-terminal kinase (JNK) but not extracellular signal-regulated kinase (ERK) or nuclear factor κB (NF-κB)
A previous study has shown that the residues lying on the switch III region and the α3 helix of Gαq are required for interaction with PLCβ [1]

Summary

Introduction

Gα16 can activate phospholipase Cβ (PLCβ) directly like Gαq. It couples to tetratricopeptide repeat 1 (TPR1) which is linked to Ras activation. Alanine scanning mutagenesis of Gαq has identified a stretch of amino acids (Ile217-Lys276) that may be responsible for PLCβ interaction. Within this region, two groups of amino acids (Asp243, Asn244, Glu245 and Arg256, Thr257; Figure 1A and 1B) have been suggested to be crucial for PLCβ interaction [1]. Two groups of amino acids (Asp243, Asn244, Glu245 and Arg256, Thr257; Figure 1A and 1B) have been suggested to be crucial for PLCβ interaction [1] These two clusters of amino acids are located in the α3 helix and β4-α3 loop (Figure 1A) which exhibits dramatic conformational changes during G protein activation [2,3]

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Discussion

Conclusion