Structural characteristics of GP and Gy subunits

GP and Gy subunits are always associated with each other as a single functional unit. Two distinct structural domains can be identified on the GP. An N-terminal helix consisting of

Table 4.1 Properties of mammalian Ga subunits

Ga subunit

Human

Cytogenetic position

cDNA genbank

Size/molecular weighta

% identity'1

Lipidationc

Phosphorylation0

Toxin sensitivit

gene

accession number

Gi Subfamily

Gaii

GNAIi

7q2i

NM_002069

3 54 a.a./40.3 kDa

i00

Myr (G2), Palm (C3)

None

PTX

Gai2

GNAI2

3p2i.3-p2i.2

J03004

355 a.a./40.5 kDa

88.i

Myr (G2), Palm (C3)

PKC, EGFR (?)

PTX

Gai3

GNAI3

ipi3

M27543

3 54 a.a./40.5 kDa

93.7

Myr (G2), Palm (C3)

None

PTX

GaoA

GNAO

i6qi2.i

AH002708

354 a.a./40.0 kDa

72.i

Myr (G2), Palm (C3)

None

PTX

Ga0B

GNAO

i6qi2.i

AH002708

3 54 a.a./40.i kDa

70.7

Myr (G2), Palm (C3)

None

PTX

Gati

GNATi

3p2i.3-p2i.2

X63749

350 a.a./40.0 kDa

67.5

Myr (G2), Others (G2)d

PKC (?)

CTX, PTX

Gat2

GNAT2

ipi3

Zi8859

3 54 a.a./40.i kDa

69.4

Myr (G2)

None

PTX

Gagust

GNAT3

7q2i

X65747 (rat)

3 54 a.a./40.i kDa

67.2

Myr (G2)

None

PTX

Gaz

GNAZ

22qii.i—qii.2

J03260

355 a.a./40.9kDa

67.0

Ara (C3), Myr (G2),

PKC (S16, 27),

None

Palm (C3)

PAK1 (S16)

Gs Subfamily

Gaolf

GNAL

i8pii.3i

U55i84

38i a.a./44.7 kDa

39.i (75.7)

Palm (C3)

None

CTX

GasL

GNAS

20qi3.2

X04408

394 a.a./45.7 kDa

38.0 (i00)

Palm (C3)

EGFR, Src (Y37,

CTX

Y377?)

GasS

GNAS

20qi3.2

X04409

380 a.a./44.2 kDa

39.i (95.9)

Palm (C3)

None

CTX

XLGas

X84047 (rat)

846 a.a./94 kDa

i7.6 (43.3)

Palm (C3)

None

CTX

Gq Subfamily

Gaq

GNAQ

9q2i

U43083

3 59 a.a./42.2 kDa

50.5 (i00)

Palm (C9, i0)

Src (Y356?)

None

Gaii

GNAii

i9pi3.3

M690i3

3 59 a.a./42.i kDa

50.0 (89.4)

Palm (C9, i0)

Src

None

Gai4

GNAi4

9q2i

NM_004297

355 a.a./4i.5 kDa

50.8 (80.2)

Palm

None

None

Ga15/16

GNAi5

i9pi3.3

M63904

374 a.a./43.5 kDa

40.9 (53.7)

Palm (C9, i0)

PKC

None

Gi2 Subfamily

Gai2

GNAi2

7p2i-p22

L0i694

379 a.a./44.i kDa

37.i (i00)

Palm (Cii)

PKC

None

Gai3

GNAi3

i7q24

NM_006572

377 a.a./44.i kDa

36.6 (63.0)

Palm (Cii)

PKC

None

a The numbers of amino acids and the corresponding molecular weights are deduced from the human open reading frames. bThe overall identities are referred to Ga^ , and identities within subfamilies are referred to the first representatives are bracketed. cThe known modification sites are shown in brackets after each type of modification. d Includes stearate, laureate, and oleate. Key: a.a.—amino acids, Ara—arachidonylation, CTX—cholera toxin, EGFR—epidermal growth factor receptor, Myr—myristoylation, PAKi—p21-activating kinase i, Palm—palmitoylation, PKC—protein kinase C, PTX—pertussis toxin, Src—Src kinase, ?—to be confirmed.

a The numbers of amino acids and the corresponding molecular weights are deduced from the human open reading frames. bThe overall identities are referred to Ga^ , and identities within subfamilies are referred to the first representatives are bracketed. cThe known modification sites are shown in brackets after each type of modification. d Includes stearate, laureate, and oleate. Key: a.a.—amino acids, Ara—arachidonylation, CTX—cholera toxin, EGFR—epidermal growth factor receptor, Myr—myristoylation, PAKi—p21-activating kinase i, Palm—palmitoylation, PKC—protein kinase C, PTX—pertussis toxin, Src—Src kinase, ?—to be confirmed.

20 residues sprouts from the core of Gp and entangles with the Gy (Fig. 4.3). The core structure of Gp is exclusively composed of antiparallel p strands arranged in seven WD repeats (Neer et al. 1994). Four p strands form a twisted p sheet and the seven sheets or blades are arranged into a propeller-like structure with the narrower side making contacts largely with the Switch II region of Ga. Gy consists of two a helices and a disordered C-terminal tail. The first helix forms a coiled-coil with the N-terminal helix of Gp subunit, whereas the second helix and the C-terminal tail extend along the wider side of the propeller core interacting with blades 5-7 of Gp (Fig. 4.3). The entire Gy practically interacts with Gp, thereby preventing their separation unless under denaturing conditions. Although direct association between GaoA and Gy2 has been reported (Rahmatullah and Robishaw 1994), there is no observable physical contact between Ga and Gy subunits in the crystal structures of trimeric Gt1 and G;1.

Accumulating evidence indicate the existence of specific combinations of Gpy complexes (Table 4.2). Much efforts have been spent on establishing the possible combinations of functional Gpy complexes using purified proteins, functional expression, yeast two-hybrid systems, or ribozyme approach (reviewed in Clapham and Neer 1997; Yan etal. 1996; Asano etal. 1999; Wang etal. 1999). The specificity of the assembly of Gpy complex appears to be determined by a stretch of 14 residues lying within the middle of Gy subunits. For Gy1, a motif of five amino acids, CCEEF, within the 14 residues are essential for specifying its interaction with Gp1. These residues mainly interact with blade 5 and the N-terminal helix of Gp.

The Gy subunit is isoprenylated at the C-terminal CAAX motif, where C is an invariant cysteine residue at —4 position of all known Gy subunits, A is aliphatic residues and X is

Table 4.2 Specificity of the assembly of Gp and Gy subunits

Gpi

GP2

GP3

GP4

GP5

GY1

+

±

-

-

-

GY2

++

++

+

+

+

gy3

+ + ++

+ + +

++

++

+ + +

gy4

+ + ++

+ + +

+

+

+ + +

gy5

+ + ++

+ + +

+

+

+

gy7

+ + ++

+ + +

+

+

±

Gyc

NR

NR

+

NR

NR

GY8

NR

NR

NR

NR

NR

GY10

+

+

-

NR

NR

GY11

+

-

-

NR

NR

GY12

NR

NR

NR

+

NR

GY13

NR

NR

+

NR

NR

Key: +, dimer formation, more plus means stronger tendencies; -, no apparent association; ±, weak interactions; NR—not reported.

Key: +, dimer formation, more plus means stronger tendencies; -, no apparent association; ±, weak interactions; NR—not reported.

undefined (Lai et al. 1990). Types 1, 11, and cone cell-specific Gy subunits are farnesylated and they all have a serine residue at the extreme C-terminus, whereas other Gy subunits having a leucine at the same position are geranylgeranylated (Table 4.3). Isoprenylation of Gy is associated with the truncation of the last three C-terminal residues and the new terminus is carboxymethylated (Casey and Seabra 1996). In addition to increasing membrane availability of GPy, specific prenyl groups may contribute to the G protein interactions with receptors and effectors. Interestingly, activation of neutrophil Gi by formyl peptide receptor is associated with carboxymethylation of Gy2, suggesting that carboxymethylation of Gy may play a role in signal transduction (Philips et al. 1995). Gy12 is the only known Gy subunit that is phosphorylated by protein kinase C at its N-terminal Ser1. Free GPy12 appears to be the preferential substrate. Phosphorylation of GPy12 selectively interferes its ability to interact with F-actin and phospholipase CP (PLCP), but not type II AC (Ueda et al. 1999 and references therein).

As observed from the crystal structures of both free GPy and GPy in heterotrimer, GP appears as a rigid body without significant conformational changes between these two states. However, subsequent resolution of a complex between GPy and phosducin indicates that GPy

Table 4.3 Properties of mammalian GpY subunits

Gp/y

Human

Cytogenetic

cDNA genbank

Size/molecular

% identityb

Covalent

subunit

gene

position

accession number

weighta

modification0

GP

GPi

GNB1

1p36.2

X04526

340 a.a./37.4 kDa

100

P, R

GP2

GNB2

7q21.1—q21.2

M16514

340 a.a./37.3 kDa

90.2

GP3

GNB3

12p13

M31328

340 a.a./37.2 kDa

83.2

GP3S

GNB3

12p13

299 a.a./32.9 kDa

74.1

GP4

GNB4

3q26-q27

AAG18442

340 a.a./37.6 kDa

90.8

GP5

GNB5

15q21

014775

353 a.a./38.6 kDa

51.2

GP5L

GNB5

15q21

AAG18444

395 a.a./43.6 kDa

45.8

G y

gyi

GNGT1

7q21.3

S62027

74 a.a./8.50 kDa

30.2

CM+F (C71)

gY2

GNG2

14q21-q22

AA868346

71 a.a./7.85 kDa

100

CM+G (C68)

gy3

GNG3

11p11

AF092129

75 a.a./8.30 kDa

72.0

CM+G (C72)

gy4

GNG4

1q43-q44

U31382

75 a.a./8.39 kDa

73.3

CM+G (C72)

gY5

GNG5

1p22

AF038955

68 a.a./7.32 kDa

47.8

CM+G (C65)

GY5-like

GNG5ps

2p12

AF188178

68 a.a./7.25 kDa

46.4

CM+G (C65)

gY7

GNG7

19p13.3

NM_005145

68 a.a./7.52 kDa

69.0

CM+G (C65)

GyC

GNGT2

17q21

014610

69 a.a./7.75 kDa

36.1

CM+G (C66)

gy8

GNG8

19q13.2—q13.3

AF188179

70 a.a./7.84 kDa

67.6

CM+F (C67)

gY10

GNG10

9q31-q32

U31383

68 a.a./7.21 kDa

53.5

CM+G (C65)

gY11

GNG11

7q21.3

U31384

73 a.a./8.48 kDa

30.2

CM+F (C70)

GY12

GNG12

1p31-p33

AF188181

72 a.a./8.01 kDa

61.6

CM+G (C69), P(S2)

GY13

GNG13

16p13.3

XM_012543

67 a.a./7.95 kDa

32.3

CM +G (C64)

a The numbers of amino acids and the corresponding molecular weights are deduced from the human open reading frames. bThe amino acid identities are referred to Gp and Gy2. cThe known modification sites are shown in blankets after each type of modification. Key: a.a.—amino acids, CM—terminal carboxymethylation, F—farnesylation, G—geranylgeranylation, P—phosphorylation, R—ADP-ribosylation.

a The numbers of amino acids and the corresponding molecular weights are deduced from the human open reading frames. bThe amino acid identities are referred to Gp and Gy2. cThe known modification sites are shown in blankets after each type of modification. Key: a.a.—amino acids, CM—terminal carboxymethylation, F—farnesylation, G—geranylgeranylation, P—phosphorylation, R—ADP-ribosylation.

can exist in two distinct conformations (Loew etal. 1998). In the 'relaxed' state, the farnesylate of Gy is exposed, mediating membrane association. In the 'tense' state, as observed in the phosducin-GpY complex, the farnesyl moiety is buried in the cavity formed between blades 6 and 7 of the Gp subunit. Binding of phosducin induces the formation of this cavity, resulting in a switch from relaxed to tense conformation, which may sequester GpY from the membrane to the cytosol and turns off the signal transduction cascade.

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