Protein Molecular Organization

The crystal structures of inactive (GDP-bound) [2], active (GTP-bound) [3-5], and transition-state (GDP-AlF4-) [4, 6] Gat, Gai, and Gas, as well as structures for inactive het-erotrimeric complexes [7,8], have provided the framework for understanding the biochemical basis of G proteins as molecular switches. In addition, comparison of Ga structures delineates the molecular basis for heterotrimeric G-protein specificity. A discussion of the specific intramolecular contacts within heterotrimeric G proteins can be found in a detailed review [9] and in another chapter of this handbook.

We currently know of 20 Ga, 5 Gp, and 12 Gy subunits. On the basis of Ga subunit similarities, G proteins are grouped into four families: Gi/o, Gs, Gq/11, and G12/13 (Table 1). Classically, members of the Gas group stimulate isoforms of adenylate cyclase (AC); however, Gas proteins also modulate the activity of other effector molecules (Table 1). The Gai/o group can be subdivided into Gai/o/z and Gat/g subunits. The former subunits inhibit some isoforms of AC but also interact with and activate other proteins (Table 1). The latter subunits stimulate the retinal cGMP-phosphodiesterase (Gat) and presumably a related gustatory effector (Gag). The Gaq/11 subunits activate the P-isoforms of phospholipase C (PLC)

Figure 1 Schematic of the GTP-binding-protein regulatory cycle. Receptor (R) interacts with a specific agonist to induce a conformational change leading to activation (R*). R* is able to interact with the heterotrimer, Ga-GDPPy and catalyze the release of GDP from the Ga subunit. Upon the binding of GTP, GPy and R* are released from Ga-GTP. Ga-GTP and free GPy regulate the appropriate effectors. The intrinsic hydrolysis of GTP to GDP, with the assistance of GTPase-activating proteins (RGS proteins) returns the system to the resting, basal state.

Figure 1 Schematic of the GTP-binding-protein regulatory cycle. Receptor (R) interacts with a specific agonist to induce a conformational change leading to activation (R*). R* is able to interact with the heterotrimer, Ga-GDPPy and catalyze the release of GDP from the Ga subunit. Upon the binding of GTP, GPy and R* are released from Ga-GTP. Ga-GTP and free GPy regulate the appropriate effectors. The intrinsic hydrolysis of GTP to GDP, with the assistance of GTPase-activating proteins (RGS proteins) returns the system to the resting, basal state.

Table I Classification of Ga Subtypes and Their Effectors

Family

Subtype

Effector

Gs

G«s(S)

T Adenylyl cyclase

G«s(L)

T GTPase of tubulina

T Adenylyl cyclase

Gi

Gai1

i Adenylyl cyclase

Gai2

Rap1 GAP

Gai3

GRIN 1 and 2

GaoA

T GTPase of tubulina

GaoB

T srca

Gaz

Ca2+ and K+ channelsa

Gat1

T cGMP-PDE

Gat2

Gag

?

Gq

Gaq

Ga11

T PLCPs

Ga14

T Bruton's tyrosine

Ga15

kinase (Gaq)

Ga16

G12

Ga12

T NH-1a

T p115RhoGEF T iNOSa

aSee references [89] to [95].

Figure 2 Hypothetical model showing the orientation of rhodopsin, the photoreceptor G protein Gt, and the membrane. The refined rhodopsin structure is from reference [98], and Gt is from reference [7]. The individual ribbon drawings are based on the crystal structures and are shown to scale. The C-terminal residues after S316 are not shown, as there is no evidence that these residues interact with Gt, and they appear to occlude the Gt binding site. The orientation of Gt with respect to rhodopsin and the membrane is modeled on the basis of the charge and hydrophobicity of the surface, the known rhodopsin-binding sites on Gt, and the sites of lipidation of Ga and Gy [7].

Figure 2 Hypothetical model showing the orientation of rhodopsin, the photoreceptor G protein Gt, and the membrane. The refined rhodopsin structure is from reference [98], and Gt is from reference [7]. The individual ribbon drawings are based on the crystal structures and are shown to scale. The C-terminal residues after S316 are not shown, as there is no evidence that these residues interact with Gt, and they appear to occlude the Gt binding site. The orientation of Gt with respect to rhodopsin and the membrane is modeled on the basis of the charge and hydrophobicity of the surface, the known rhodopsin-binding sites on Gt, and the sites of lipidation of Ga and Gy [7].

and nonreceptor protein kinases of the 5iMamily (Bruton's tyrosine kinase). The Ga12/13 subunits regulate the activity of low-molecular-weight G proteins of the Rho family, the Na+/H+-exchanger, phospholipase D (PLD) and the inducible isoform of NO synthase (iNOS).

All Ga subunits are formed by two domains: a GTPase domain that is involved in binding and hydrolysis of GTP and a helical domain that buries the guanine nucleotide within the core of the protein (Fig. 2). The helical domain is the most divergent domain among Ga families and may have a role in directing specificity of interaction with partner proteins. In the GTPase domain, three nonconsecutive flexible regions undergo structural change between the GDP-and GTP-bound conformations [2,3]. These regions, designated Switch I, II and III, become more rigid and well ordered in the GTP-bound conformation. The structures of the extreme amino (N)- and carboxyl (C)-terminal domains are not solved in the isolated Ga crystals, as the N- and C-termini were either removed from the protein or disordered [2-4].

However, in two separate crystal structures of heterotrimeric complexes, the N-terminal helix is ordered by its interaction with GP [7,8].

Crystallographic studies of GPy heterodimer alone [10] and in the heterotrimeric complex [7,8] revealed that the GP subunit has a P-propeller structure containing 7 WD-40 repeats (Fig. 2). The Gy subunit interacts with the GP subunit through an N-terminal coiled-coil and makes extensive contacts along the base of the GP subunit. GP, but not Gy, contacts Ga in two regions, the N-terminal helix and a hydrophobic pocket present in Ga-GDP that includes the switch II region [7,8].

Diabetes Sustenance

Diabetes Sustenance

Get All The Support And Guidance You Need To Be A Success At Dealing With Diabetes The Healthy Way. This Book Is One Of The Most Valuable Resources In The World When It Comes To Learning How Nutritional Supplements Can Control Sugar Levels.

Get My Free Ebook


Post a comment