Extended Data Table 1.

GRK mutations and their impact on kinase activity towards Rho* or a soluble substrate.

αN Helix		Rho*	soluble substrate	Ref
ΔN19	GRK1	---- †	---	29
ΔN2–14	GRK5	----	NC‡	7
D2A	GRK1	NC	NC	62
D3A	GRK2	-	ND§	5,34
D3N	GRK2	-	ND	34
D3K	GRK2	----	NC	34
S5E	GRK1	+||	--	¶
S5A	GRK1	--	ND	45
L6A	GRK1	---	+	35
L4A	GRK2	---	NC	34
L4A	GRK2	--	+	5
L4K	GRK2	----	ND	34
L3Q	GRK5	--	ND	7
L3Q/K113R	GRK5	---	NC	7
L3A	GRK6	-	NC	6
E7A	GRK1	---	+	35
E5A	GRK2	-	NC	34
E4A	GRK6	-	-	6
T8A	GRK1	-	ND	45
T8E	GRK1	--	ND	45
V9A	GRK1	----	+	35
V7E	GRK2	--	+	5
I6A	GRK6	---	NC	6
V10A	GRK1	----	+	35
V7A	GRK6	---	NC	6
L7A/8A	GRK2	----	ND	34
I6A/V7A	GRK6	----	NC	6
I6E/V7E	GRK6	----	NC	6
A11E	GRK1	-	-	35
N12A	GRK1	----	---	35
D10A	GRK2	---	NC	5,34
D10A	GRK2	-	-	5
D10R	GRK2	--	-	5
N9A	GRK6	----	--	6
T10P	GRK5	--	NC	7
F15A	GRK1	----	----	35
Y13A	GRK2	----	-	5
L12A	GRK6	----	-	6
I16A	GRK1	--	--	35
L13A	GRK6	--	--	6
M15A	GRK2	NC	+	5
A18E	GRK1	--	--	35
A16V	GRK2	NC	-	5
M17A	GRK2	-	-	5
E18A	GRK2	+	ND	5
AST region		Rho*	soluble substrate	Ref
P473A	GRK2	NC	ND	63
P473E	GRK2	-	-	63
R474A	GRK2	NC	ND	5
R470A	GRK5	-	ND	64
G475I	GRK2	-	-	5
E476K	GRK2	NC	ND	63
V476A	GRK1	----	---	29
V477D	GRK2	---	-	63
N478A	GRK2	NC	ND	5
A479S	GRK2	NC	ND	5
A480S	GRK2	NC	ND	5
N480A	GRK1	+	ND	29
D481A	GRK2	NC	ND	63
A482I	GRK2	-	ND	5
Q482A	GRK1	+	ND	29
F483A	GRK2	NC	ND	63
D483A	GRK1	-	ND	29
D484A	GRK2	NC	ND	63
V484A	GRK1	----	----	29
I485A	GRK2	-	-	63
F487A	GRK1	-	ND	29
F488D	GRK2	NC	ND	63
S488E/T489E	GRK1	NC	-	¶
S488D/T489D	GRK1	-	-	¶
S5E/S488E/T489E	GRK1	+	NC	¶
E490K	GRK2	NC	ND	63
G495A	GRK2	NC	ND	63
L499D	GRK2	NC	ND	63
Small lobe		Rho*	soluble substrate	Ref
F190A	GRK1	---	----	29
F190Y	GRK1	NC	ND	29
F190H	GRK1	+	ND	29
R191A	GRK1	----	----	29
R191K	GRK1	----	----	29
R195A	GRK2	----	ND	29
R190A	GRK6	----	ND	29
V192A	GRK1	NC	ND	29
R195A	GRK1	-	ND	29
Q205A	GRK1	+	ND	29
G210E	GRK1	--	ND	29
L212A	GRK1	---	---	29
L212M	GRK1	+	ND	29
N268A	GRK1	NC	ND	29
Large lobe		Rho*	soluble substrate	Ref
H280A	GRK2	NC	ND	63
Y274A	GRK1	----	----	29
Y274F	GRK1	+	ND	29
Y281A	GRK2	-	-	63
H275A	GRK1	+	ND	29
S284A	GRK2	NC	ND	63
Q285A	GRK2	NC	ND	63
Q285K	GRK2	NC	ND	63
K395A	GRK2	NC	ND	63
K397A	GRK2	NC	ND	63
RH domain		Rho*	soluble substrate	Ref
5A	GRK1	----	ND	¶
D164A	GRK1	-	ND	45
L166K	GRK1	-	ND	45
D164A/L166K	GRK1	-	ND	45
L33N	GRK2	NC	NC	2
E36A	GRK2	NC	NC	65,2
V42E	GRK2	--	ND	65
Y46A	GRK2	-	ND	65
R68A	GRK5	+	ND	66
P61A/Q69A	GRK5	=	ND	66
L66A/H38A	GRK5	---	ND	66
L66A/P73A	GRK5	---	ND	66
L66A/E514A	GRK5	---	ND	66
L66A/P37A	GRK5	---	ND	66
L66A/R69A	GRK6	--	ND	66
L66A/Q172A	GRK6	----	ND	66
F166A/H38A	GRK5	---	ND	66
F166A/E514A	GRK5	---	ND	66
F166A/P61A	GRK5	---	-	66
F166A/L66A	GRK5	---	-	66
Y166A/L66A	GRK6	----	ND	66
F166A/Q172A	GRK5	---	ND	66
Y166A/Q172A	GRK6	----	ND	66
Y166A/L176A	GRK6	---	ND	66
F166A/W173A	GRK5	---	-	66
Q172A/I176A	GRK5	---	-	66
Q172A/W173A	GRK6	----	ND	66
E96A	GRK2	NC	ND	65
R516A	GRK2	NC	ND	65
E520A	GRK2	--	ND	65
E532A	GRK2	NC	NC	2
L536N	GRK2	NC	NC	2
L547N	GRK2	NC	NC	2
P638D	GRK2	--	ND	65
V92A	GRK5	ND	NC	31
V92L	GRK5	ND	NC	31
V92M	GRK5	ND	NC	31
K454A	GRK5	ND	NC	31
R455A	GRK5	ND	NC	31
K454A/R455A	GRK5	ND	NC	31
I39E/I165E	GRK6	NC	ND	67
I165E/F527D	GRK6	NC	ND	67

^†----, >100-fold reduction; ----, 20–100 fold reduction; ---, 10–20 fold reduction; --, 5–10 fold reduction; -, 3–5 fold reduction.

^‡NC, no change.

^§ND, not determined.

^||+, 1–3 fold increase.

^¶Mutation is characterized in the current paper.