The β6/α5 regions of Gαi2 and GαoA increase the promiscuity of Gα16 but are insufficient for pertussis toxin-catalyzed ADP-ribosylation

Characterization of CHO Cells Stably Expressing a Gα16/zChimera for High Throughput Screening of GPCRs

“…11,14 However, as G␣ 16 cannot couple to all GPCRs, 12,17 we have developed and characterized chimeric proteins based on G␣ 16 that incorporate elements of G␣ z , G␣ i , G␣ o , and G␣ s proteins. [18][19][20] Of particular interest are the 16z25 and 16z44 chimeras, which incorporate the C-terminal 25 and 44 amino acids of G␣ z into G␣ 16 (Fig. 1).…”

“…17 The same study also demonstrated that the G i/o -coupled ␦-opioid, -opioid, and N-formylmethionyl-leucyl-phenylalanine receptors similarly do not promote Ca 2ϩ release in the absence of a chimera, and we have subsequently confirmed the PTX insensitivity of PLC␤-mediated responses in cells expressing these receptors and 16z44. 20 This indicates that G i/o -coupled receptors that are able to promote Ca 2ϩ release in the presence of a chimera are not signaling via endogenous G i/o proteins. SST 2 receptors weakly mediate inositol phosphate production in transfected Chinese hamster lung fibroblast cells, 31 and only modestly promote Ca 2ϩ responses in COS-7 cells, but the latter response can be potentiated almost fivefold in the presence of 16z25.…”

“…1), 18 as well as a range of chimeras incorporating elements of G␣ s and G␣ i/o . 19,20 These chimeras have proven to recognize Ͼ20 G i/o -and G s -coupled receptors in both FLIPR and reporter gene-based assays using cells transiently coexpressing the receptors and chimeric G proteins. At least 12 of those GPCRs do not demonstrate a substantial functional interaction with G␣ 16 .…”

“…At least 12 of those GPCRs do not demonstrate a substantial functional interaction with G␣ 16 . [17][18][19][20][21] To facilitate the use of these chimeric G proteins in high throughput FLIPR assays, we now describe the generation and characterization of cell lines stably expressing 16z25 and one of nine different GPCRs. 16 (81482) was purchased from Gramsch Laboratories (Schwabhausen, Germany).…”

Identification of a stretch of six divergent amino acids on the alpha5 helix of Galpha16 as a major determinant of the promiscuity and efficiency of receptor coupling

“…This peculiarity of Gα 16 has been investigated by manipulating its amino acid sequence in the receptor-coupling domains [11][12][13]. Swapping the last 25 or 44 amino acids of Gα 16 with those of Gα z , Gα i2 and Gα o allows the chimaeric Gα subunits to couple with more G i -linked receptors [9,14,15]. The same strategy has also been applied for redirecting the coupling specificity of Gα 16 towards G s -coupled receptors [16].…”