ABSTRACT: BACKGROUND: Galpha16 can activate phospholipase Cbeta (PLCbeta) directly like Galphaq. It also couples to tetratricopeptide repeat 1 (TPR1) which is linked to Ras activation. It is unknown whether PLCbeta and TPR1 interact with the same regions on Galpha16. Previous studies on Galphaq have defined two minimal clusters of amino acids that are essential for the coupling to PLCbeta. Cognate residues in Galpha16 might also be essential for interacting with PLCbeta, and possibly contribute to TPR1 interaction and other signaling events. RESULTS: Alanine mutations were introduced to the two amino acid clusters (246-248 and 259-260) in the switch III region and alpha3 helix of Galpha16. Regulations of PLCbeta 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 Galpha16 was completely abolished by mutating either clusters. Contrastingly, phosphorylations of extracellular signal-regulated kinase (ERK) and nuclear factor kappaB (NF-kappaB) were not significantly affected by these mutations. The interactions between the mutants and PLCbeta2 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 alpha3 helix. Mutations of both clusters almost completely abolished the receptor coupling and prevent receptor-induced Gbetagamma release. CONCLUSION: The integrity of the switch III region and alpha3 helix of Galpha16 is critical for the activation of PLCbeta, STAT3, and JNK but not ERK or NF-kappaB. Binding of Galpha16 to PLCbeta2 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.