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 G_q 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 G_q (Gα_q) 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 G_q family. The PLC-β interacting regions on Gα_q had been mapped out in a previous study. By amino acid sequence alignment, two groups of potentially PLC-β inte...[ Read more ]

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 G_q 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 G_q (Gα_q) 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 G_q family. The PLC-β interacting regions on Gα_q 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 Gα_q. (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 G_i or G_s-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.