||Heterotrimeric guanosine triphosphate-binding proteins (G proteins) serve as both the upstream initiators of different second messengers and the downstream targets of the various heptahelical transmembrane receptors. Members of the Gq subfamily of heterotrimeric G proteins regulate the β-class of phosphoinositide-specific phospholipase C (PLC-β). In the current understanding, most downstream effectors of the α subunit of Gq (Gαq) are triggered through the activation of PLC-β. Hence, constructing defective PLC-β activating mutants can be used to study the signaling cascade triggered by Gα16, a hematopoietic-specific member of the Gq family. The PLC-β interacting regions on Gαq had been mapped out in a previous study. By amino acid sequence alignment, two groups of potentially PLC-β interacting amino acids were found on Gα16. Residues 246-248 are next to the switch III region and very close to the receptor interacting region, and residues 259 and 260 are located in the α3 helix which is close to the carboxyl terminus. Using alanine replacement mutagenesis, the PLC-β interacting ability of point mutated mutants will be knocked down completely or partially. The objectives of the present study are to investigate (1) the function of selected amino acid residues on Gα16 which are responsible for PLC-β interaction on Gαq. (2) the role of PLC-β in Gα16 elicited signaling pathway. The results suggested that mutations at either residues Asn245-Glu247 or Gly259-Thr260 lowered the affinity for PLC-β but could not completely suppress the activation of PLC-β. In other words, Asn245-Glu247 and Gly259-Thr260 are not the only regions responsible for PLC-β interaction. Other downstream effectors of Gα16 such as JNK, STAT3, NF-κB and ERK were similarly affected. Results also revealed that substitution of Asn245-Glu247 and Gly259-Thr260 by alanine led to the disruption of receptor interacting ability. Replacement of either Asn245-Glu247 or Gly259-Thr260 diminishes Gi or Gs-coupled receptors induced PLC-β responses by 50%-80%. In summary, amino acid clusters Asn245-Glu247 and Gly259-Thr260 appear to play critical roles in receptor interaction in additional to their regulation of PLC-β.