Gα16 interacts with tetratricopeptide repeat 1 (TPR1) through its β3 region to activate Ras independently of phospholipase Cβ signaling

Table 3 Gα_16/z chimera-mediated phosphorylations of STAT3 (both Tyr⁷⁰⁵ and Ser⁷²⁷), IKK, and ERK in HEK 293 cells.

*Construct*	*TPR1/Ras Interaction*	*PLCβ*	*Fold Stimulation of Protein Phosphorylation*
		*Activity*	*P-Tyr* ⁷⁰⁵ *STAT3*	*P-Ser* ⁷²⁷ *STAT3*	*P-IKK*	*P-ERK*
pcDNA1	N/A	N/A	1.0 ± 0.0	1.0 ± 0.0	1.2 ± 0.1	1.1 ± 0.1
Gα₁₆	Yes	Yes	2.3 ± 0.3*	2.0 ± 0.1*	2.4 ± 0.3*	2.6 ± 0.1*
N30	Yes	Yes	1.7 ± 0.1*	1.7 ± 0.2*	1.7 ± 0.2*	1.9 ± 0.1*
N102	Yes	No	1.2 ± 0.1	1.1 ± 0.1	1.2 ± 0.0	1.3 ± 0.1
N155	Yes	No	1.1 ± 0.0	1.1 ± 0.1	1.2 ± 0.0	1.5 ± 0.0
N188	Yes	Yes	1.7 ± 0.0*	1.4 ± 0.1*	1.9 ± 0.2*	2.2 ± 0.1*
N200	Yes	Yes^a	0.9 ± 0.0	1.1 ± 0.1	1.6 ± 0.2*	1.9 ± 0.1*
N210	No	No	0.9 ± 0.1	0.9 ± 0.0	1.0 ± 0.0	1.1 ± 0.0
N246	No	No	1.1 ± 0.1	1.1 ± 0.1	1.0 ± 0.0	1.1 ± 0.1
N266	No	No	1.4 ± 0.3	1.1 ± 0.0	1.2 ± 0.2	1.3 ± 0.1
N295	No	No	1.0 ± 0.1	0.9 ± 0.0	1.0 ± 0.1	1.1 ± 0.0
C25	Yes	Yes	1.7 ± 0.1*	1.5 ± 0.1*	1.6 ± 0.1*	2.2 ± 0.0*
C44	Yes	Yes	1.6 ± 0.0*	1.4 ± 0.0*	1.5 ± 0.1*	1.8 ± 0.0*
C164	Yes	No	0.9 ± 0.1	1.0 ± 0.1	1.0 ± 0.0	1.3 ± 0.0
C174	No	No	1.1 ± 0.1	0.9 ± 0.0	0.6 ± 0.1	1.2 ± 0.2
C186	No	No	0.9 ± 0.1	1.1 ± 0.2	1.0 ± 0.1	1.3 ± 0.1
C219	No	No	1.1 ± 0.1	1.1 ± 0.2	1.3 ± 0.2	1.2 ± 0.1
C272	No	No	1.0 ± 0.1	0.9 ± 0.1	1.1 ± 0.1	0.8 ± 0.0
zβ2β3	No	Yes	1.6 ± 0.1*	1.5 ± 0.1*	1.5 ± 0.1*	2.2 ± 0.2*
zβ3	No^b	Yes	2.1 ± 0.2*	1.9 ± 0.1*	2.0 ± 0.2*	2.7 ± 0.1*
N200-C164	Yes	No	1.0 ± 0.1	1.1 ± 0.1	0.9 ± 0.1	1.2 ± 0.0
N188-C164	Yes	No	1.0 ± 0.0	0.9 ± 0.1	1.0 ± 0.1	1.2 ± 0.0
Gα_z	No	No	1.0 ± 0.0	0.9 ± 0.0	1.1 ± 0.0	1.1 ± 0.2

HEK 293 cells were co-transfected with adenosine A₁R and pcDNA1 or the indicated Gα subunit. Transfectants were serum starved overnight in the presence of 100 ng/mL of PTX and then challenged with 10 μM CHA for 15 min. Cell lysates were then resolved and immunoblotted against various specific antibodies. Results are expressed as fold stimulation over the basal (DMSO vehicle), n = 3; significant responses are shown in bold and italic. Constructs shown to co-immunoprecipitate with FLAG-TPR1 or stimulate PLCβ activity (Table 2) are marked with a "Yes".
* CHA-stimulated phosphorylation of the target protein is significantly greater than the basal; paired t-test, p ≤ 0.05.
^asignificant PLCβ activation observed with the QL mutant only (see Table 2).
^bVery weak association with TPR1 was detected in co-immunoprecipitation assays (Figure 9).

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