Substituted by cognate residues from Gz. Interestingly, an intact N-terminal helical domain (A-F) of G14 are seemingly important for G14-mediated PLC activation. G14 chimeras with A-F replaced entirely or in part by Gz can be expressed at a detectable level but failed to interact with PLC2 or stimulate IP3 Lenvatinib production.Discussion Structure and function correlations of members within the same protein family are often based on extensive analyses of a prototypical member. In the case of the Gq family, it is generally assumed that all members interact with the canonical effector PLC in much the same way as Gq. The biochemical functions of Gq family members are almost indistinguishable [33, 37] except for their ability to recognize specific receptors [38]. Hence, it is rather surprising that the putative PLC-interacting domains identified from studies on Gq [13, 14, 17, 39] are simply insufficient to support efficient regulation of PLC by G14. Although detailed structural comparison between Gq and G14 is not feasible because of the lack of G14 structural data, the overall sequence similarity of over 80 indicates a highly conserved three-dimensionalKwan et al. BMC Structural Biology (2015) 15:Page 8 ofABCFig. 5 Role of the N-terminal helix (N) in the Gq-PLC3 complex. a The model of Gq (light orange) is shown as a space filling structure and contains the N-helix and other regions as indicated. PLC3 (yellow) is depicted as a cartoon ribbon, containing the helix-turn-helix segment (H1/H2), the N-terminal PH domain, four EF hands, the catalytic TIM barrel, and a C2 domain. PLC3-interacting residues of Gq are colored in magenta. The carboxy-terminal (CT) domain of PLC3 is not included in the structural model. The structure of the N-helix is generated by replacing the amino acid sequence of Gi (Gi12, PDB code: 1GP2) with the Gq sequence. The final model is generated by alignment of Gq-PLC3 (PDB code: 3OHM) and the modified heterotrimer Gq12 using PyMOL (The PyMOL Molecular Graphics System, Version 1.3 Schr inger, LLC). The orientation of the N-helix represents the conformation in the heterotrimer and is not optimized for the Gq-PLC3 complex. In this case, the N-helix points towards the cell membrane and clashes with PLC3, but in fact may exist in a conformation which interacts with PLC3. b Schematic representation of 14 and zN chimeras. c Cells were co-transfected with PLC2 and G protein or the indicated chimeras. Co-immunoprecipitation assays were performed and analyzed as in Fig. 2. Data shown represent one of three sets of PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/8627573 PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/22316373 immunoblots; two other sets yielded similar results. For the IP3 accumulation assay, HEK293 cells were transiently transfected with the wild-type or constitutively active mutants (QL) of G proteins or chimeras and analyzed as in Fig. 2. *, IP3 production was significantly enhanced as compared to corresponding wild-type transfected cells; Dunnett t test, p < 0.structure shared by both proteins [18, 19]. Since the structural homology and the residues responsible for PLC interaction and activation are presumably conserved from Gq to G14, one would expect that G14 may utilize the same residues for PLC activation. It should also be noted that sequence variations in interacting residues of PLC2 and PLC3 may affect the ability of G14 to efficientlystimulate PLC2. In particular, conservative substitutions such as D973E and Q1066S in the distal CTD may have limited consequences for G14 binding, whereas more severe mutations in other in.