A, the DEF region of G14 seems necessary for interaction with TPR1. However, several chimeras lacking this region, including 182z14, 203z14, and 131z14, are able to interact with TPR1. The inability of the zDEF chimera to interact with TPR1 could be caused by disruption of the helical domain resulting in instability of the protein structure. Disruption of the GTPase domain and substitution of the helical domain by Gz , as demonstrated by the z243 chimera, did not completely abolish TPR1 interaction. Moreover, the mirror image pairs 14z151 and 203z14, as well as 14z173 and 182z14, were able to interact with TPR1. These results indicate that the presence of either the 2-4-3 region of the GTPase domain or an intact helical domain of G14 is sufficient for TPR1 interaction.Conclusion The present study has successfully used chimeric G14/ Gz constructs to map critical regions for effector regulation and demonstrates the insufficiency PRIMA-1 of previous structural information in supporting efficient effector regulation by G proteins. Although the roles of the N helix and helical domain of G subunits in G proteinmediated signal transduction have mostly been neglected, our results designate important roles for these domains of G14 in effector interaction and activation. MethodsReagentsThe human cDNAs of G14, G14QL were obtained from Guthrie Research Institute (Sayre, PA, USA). Cell culture reagents, including LipofectAMINE PLUS reagents, and Lipofectamin 2000 were purchased from Invitrogen (Carlsbad, CA, USA). Anti-G14 targeting the N-terminal was obtained from Gramsch Laboratories (Schwabhausen, Germany). Anti-FLAG antibody and anti-FLAG affinity gel were from Sigma-Aldrich (St. Louis, MO, USA). Other antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Protein G-agarose and dithiobis[succinimidylpropionate] (DSP) cross-linker were from Pierce Biotechnology (IL, USA). Osmonics nitrocellulose membrane and ECL kit were from Westborough (MA, USA) and Amersham (Piscataway, NJ, USA), respectively. Pertussis toxin (PTX) was obtained from List Biological Laboratories (Campbell, CA, USA), and octreotide (OCT) was from Sigma-Aldrich (St. Louis, MO, USA).Kwan et al. BMC Structural Biology (2015) 15:Page 12 ofCell culture and co-immunoprecipitationHEK293 cells were obtained from the American Type Culture Collection (CRL-1573, Rockville, MD). They were maintained in Eagle's minimum essential medium at 5 CO2, 37 with 10 fetal bovine serum, 50 units/mL penicillin and 50 g/mL streptomycin. For coimmunoprecipitation experiments, HEK293 cells were grown to 80 confluency in 100 mm tissue culture plates and then co-transfected with 200 ng G and 200 ng FLAG-TPR1 cDNAs using 15 L PLUS and LipofectAMINE reagents in Opti-MEM. Serum was replenished 3 h after transfection. Cross-linking was performed one day after transfection; transfected HEK293 cells were washed with PBS twice and then treated with 0.5 mM DSP in PBS for 15 min at room temperature. Cells were then washed again with PBS and maintained in quenching solution (50 mM glycine in PBS, pH 7.4) for PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/18218841 5 min. Subsequently, cells were lysed in ice-cold RIPA buffer (25 mM HEPES at pH 7.4, 0.1 SDS, 1 Nonidet P-40, 0.5 sodium deoxycholate, 1 mM dithiothreitol, 200 M Na3VO4, 4 g/mL aprotinin,100 M phenylmethylsulfonyl fluoride, and 2 g/mL leupeptin). Cell PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/9547713 lysates were gently rocked with an anti-G14 antiserum at 4 overnight, and then incubated in 30 L protein G-agarose (50 slurry) at 4.