**3. Trimeric G proteins**

#### **3.1 G protein subtypes and GPCR effectors**

The G proteins that transduce the signal from GPCRs are heterotrimeric (Gαβγ) proteins. Some of the mRNAs encoding the Gα subunits are subject to alternative splicing so that sixteen genes encode 23 known Gα proteins: (Birnbaumer, 2007). The Gα proteins are anchored to the plasma membrane by N-terminal myristoylation or palmitoylation. They can be grouped into four families based upon sequence homologies, and each GPCR has a preference for a single Gα or for a single family of Gα subunits. Each Gα subunit regulates one or a few effectors (Birnbaumer, 2007):


The carboxyl-terminal Gα sequence is the major determinant for receptor recognition: exchanging this sequence allows the construction of promiscuous chimeric G proteins that can be used to drive GPCR coupling to a non-physiological effector (Kostenis *et al.*, 2005).

There are five known human Gβ and 12 Gγ genes (Birnbaumer, 2007). Most but not all of the Gβγ and Gα- Gβγ combinations are allowed. All Gγ subunits are C-terminally prenylated (some with geranyl-geranyl, others with farnesyl groups) and carboxymethylated: this helps to anchor the Gβγ subunits to the plasma membrane. The C-terminal sequence determines the nature of the prenyl group (farnesyl or geranyl-geranyl) modifying the Gγ subunit; both the C-terminal sequence and the prenyl group play an active role in the recognition of both rhodopsin and phospholipids (Katadae *et al.*, 2008). Although the literature on this subject is sparse, there is some evidence that other GPCRs also recognize preferentially specific Gβγ subunits (Jian *et al.*, 2001; Kisselev and Downs, 2003; Birnbaumer, 2007). The Gβγ subunits recognize and regulate a growing list of effectors, including ion channels, phospholipase C (PLC), phosphoinositide-3' kinase-γ (PI3Kγ), various adenylate cyclase isoforms, etc. Different PLC isoforms respond differently to different Gβγ isoforms; and the cardiac ATPinhibited inwardly rectifying K+ channel (KirATP) is either inhibited or activated by Gβγ depending on the nature of the Gβ subunit (Birnbaumer, 2007).

### **3.2 G proteins as (inefficient) GTPases**

G proteins are (poor) GTPases (Birnbaumer, 2007): they hydrolyze GTP slowly to GDP + inorganic phosphate, then release GDP extremely slowly. The GDP release and the GTP hydrolysis reactions are highly regulated, accompanied by conformation changes, and used as molecular clocks.

Trimeric G proteins are no exception to this rule: GTP binding is necessary to allow transient effectors activation (Oldham and Hamm, 2006; Birnbaumer, 2007). As summarized in Figure 2, the GDP release from trimeric G proteins is accelerated by G Protein Coupled Receptors (GPCRs) that function as "Guanyl nucleotides Exchange Factors" (GEFs): they allow GDP release, and this is rapidly followed by GTP recognition and dissociation of the two G protein subunits. Both subunits can then transiently recognize their respective effectors. The GTP hydrolysis reaction (leading to signal interruption) is accelerated by "Regulators of G

sixteen genes encode 23 known Gα proteins: (Birnbaumer, 2007). The Gα proteins are anchored to the plasma membrane by N-terminal myristoylation or palmitoylation. They can be grouped into four families based upon sequence homologies, and each GPCR has a preference for a single Gα or for a single family of Gα subunits. Each Gα subunit regulates

G proteins in the Gi (Gi/o/t/gust/z) G protein family inhibit adenylate cyclase and/or

 G proteins in the G12/13 G protein family activate "Guanyl nucleotide Exchange Factors" (GEFs) that in turn activate another group of "small" (monomeric) G proteins,

The carboxyl-terminal Gα sequence is the major determinant for receptor recognition: exchanging this sequence allows the construction of promiscuous chimeric G proteins that can be used to drive GPCR coupling to a non-physiological effector (Kostenis *et al.*, 2005).

There are five known human Gβ and 12 Gγ genes (Birnbaumer, 2007). Most but not all of the Gβγ and Gα- Gβγ combinations are allowed. All Gγ subunits are C-terminally prenylated (some with geranyl-geranyl, others with farnesyl groups) and carboxymethylated: this helps to anchor the Gβγ subunits to the plasma membrane. The C-terminal sequence determines the nature of the prenyl group (farnesyl or geranyl-geranyl) modifying the Gγ subunit; both the C-terminal sequence and the prenyl group play an active role in the recognition of both rhodopsin and phospholipids (Katadae *et al.*, 2008). Although the literature on this subject is sparse, there is some evidence that other GPCRs also recognize preferentially specific Gβγ subunits (Jian *et al.*, 2001; Kisselev and Downs, 2003; Birnbaumer, 2007). The Gβγ subunits recognize and regulate a growing list of effectors, including ion channels, phospholipase C (PLC), phosphoinositide-3' kinase-γ (PI3Kγ), various adenylate cyclase isoforms, etc. Different PLC isoforms respond differently to different Gβγ isoforms; and the cardiac ATPinhibited inwardly rectifying K+ channel (KirATP) is either inhibited or activated by Gβγ

G proteins are (poor) GTPases (Birnbaumer, 2007): they hydrolyze GTP slowly to GDP + inorganic phosphate, then release GDP extremely slowly. The GDP release and the GTP hydrolysis reactions are highly regulated, accompanied by conformation changes, and used

Trimeric G proteins are no exception to this rule: GTP binding is necessary to allow transient effectors activation (Oldham and Hamm, 2006; Birnbaumer, 2007). As summarized in Figure 2, the GDP release from trimeric G proteins is accelerated by G Protein Coupled Receptors (GPCRs) that function as "Guanyl nucleotides Exchange Factors" (GEFs): they allow GDP release, and this is rapidly followed by GTP recognition and dissociation of the two G protein subunits. Both subunits can then transiently recognize their respective effectors. The GTP hydrolysis reaction (leading to signal interruption) is accelerated by "Regulators of G

G proteins in the Gs (Gs/olf) G protein family stimulate adenylate cyclase,

depending on the nature of the Gβ subunit (Birnbaumer, 2007).

**3.2 G proteins as (inefficient) GTPases**

as molecular clocks.

G proteins in the Gq/11 (Gq/11/14/15/16) G protein family activate phospholipase C,

one or a few effectors (Birnbaumer, 2007):

regulate ion channels,

the Rho G proteins

protein Signaling" (RGS) proteins, that function as "GTPase Activator Proteins" (GAPs) and accelerate signal interruption.

Fig. 2. the G protein activation cycle. In the resting state, the G protein is trimeric (GαGDPGβγ) and occupied by GDP. GPCRs interact with resting (GDP-bound) G proteins, facilitate the GDP release and stabilize an empty G protein conformation. GTP induces the dissociation of the two G protein subunits, GαGTP and Gβγ: this allows both subunits to recognize and regulate their respective effectors (enzymes, channels or regulators of G protein signaling). GαGTP hydrolyses GTP to GDP and the GαGDP complex recognizes Gβγ with a very high affinity: the resting trimeric complex reforms spontaneously. GTP hydrolysis can be accelerated by "Regulators of G protein Signaling" (RGS) molecules.

## **3.3 G protein structures**

The G proteins regulated by GPCRs are heterotrimeric (Birnbaumer, 2007). The three polypeptide chains form two independent subunits: the Gα and Gβγ subunits (Figure 3). The Gα protein structure can be divided into two domains held together by mutual interactions with the guanyl nucleotide: a N-terminal "Ras-like domain" (with strong structural homology with the small GTPases, Ras) and a C-terminal α-helical domain (Figure 3). In the agonist-receptor-G protein complex, the guanyl nucleotide has dissociated, and the helical domain "floats away" from the Ras –like domain (Rasmussen *et al.*, 2011b). The Gβ protein "WD repeats" (blue) forms a 7 blades beta-propeller domain, and the Gγ protein (green) wraps around Gβ, one of the two small α-helices forming a coiled-coil with the Gβ protein α-helix (Figure 3). Gβγ forms a stable complex that cannot be dissociated without denaturation but Gα can dissociate from Gβγ upon GTP binding.

As shown in Figure 4, the conformation of three segments ("switch regions") of Gα changes during the GTPase catalytic cycle (Oldham and Hamm, 2006; Rasmussen *et al.*, 2011b). This regulates the interaction of Gα with Gβγ and with its effectors. Switch 2 together with either switch 1 or 3 indeed forms part of the Gα – protein binding interface (Figure 5). It participates to the recognition of Gβγ, but also of effectors and "Regulators or G protein Signaling" (RGS) proteins (Figure 5): the Gα- Gβγ dissociation is essential to allow effectors activation by GαGTP.

Fig. 3. Ribbon representations of G protein structures. The Gα subunits are presented in yellow, the β subunits in blue and the γ subunits in green and the β2-adrenergic receptor, in red. GDP and GTP are shown in fuchsia as space-filling structures. From left to right: the Gi protein (1GG2), the β2 adrenergic receptor-agonist-Gscomplex (3SN6), the activated GαGTP (1GIL) and Gβ1γ2 (1TBG) structures. The latter two structures have been rotated separately by approximately 90° compared to GαGDPβγ, to show the different domains (center right: the ras-like and α-helical domains of the Gα subunit; far right: a β-propeller structure in the Gβ subunit).

Fig. 4. Effect of guanyl nucleotide binding on the Gα subunit conformation. Ribbon representation of Gα in different crystallized complexes : the Gα subunit only is shown for simplicity; the Gβγ subunit when present would be in front and to the right of the Gα subunit. The N-terminal region, when structurally defined (stabilized by interactions with Gβγ) is represented by a blue ribbon. Three regions change conformation during the GTPase catalytic cycle: "switch 1" is shown in green, "switch 2" in gold, and "switch 3" in red. GDP (pink), GTP (yellow) and phosphate (pink) are shown as space filling structures. Left: the Gα<sup>t</sup> subunit in the GαGDPβγ complex (PDB 1GOT); top center: the structure of Gα<sup>S</sup> in the ternary complex, HRG (PDB 3SN6), stabilized by a nanobody; right: the structure of the GTP analogue GTPγS-activated Gα<sup>t</sup> (PDB 1TND) and bottom center: the structure of Gα<sup>i</sup> bound to GDP and inorganic phosphate during GTP hydrolysis (PDB 1GIT).

Fig. 3. Ribbon representations of G protein structures. The Gα subunits are presented in yellow, the β subunits in blue and the γ subunits in green and the β2-adrenergic receptor, in red. GDP and GTP are shown in fuchsia as space-filling structures. From left to right: the Gi protein (1GG2), the β2 adrenergic receptor-agonist-Gscomplex (3SN6), the activated GαGTP (1GIL) and Gβ1γ2 (1TBG) structures. The latter two structures have been rotated separately by approximately 90° compared to GαGDPβγ, to show the different domains (center right: the ras-like and α-helical domains of the Gα subunit; far right: a β-propeller

Fig. 4. Effect of guanyl nucleotide binding on the Gα subunit conformation. Ribbon representation of Gα in different crystallized complexes : the Gα subunit only is shown for simplicity; the Gβγ subunit when present would be in front and to the right of the Gα subunit. The N-terminal region, when structurally defined (stabilized by interactions with Gβγ) is represented by a blue ribbon. Three regions change conformation during the GTPase catalytic cycle: "switch 1" is shown in green, "switch 2" in gold, and "switch 3" in red. GDP (pink), GTP (yellow) and phosphate (pink) are shown as space filling structures. Left: the Gα<sup>t</sup> subunit in the GαGDPβγ complex (PDB 1GOT); top center: the structure of Gα<sup>S</sup> in the ternary complex, HRG (PDB 3SN6), stabilized by a nanobody; right: the structure of the GTP analogue GTPγS-activated Gα<sup>t</sup> (PDB 1TND) and bottom center: the structure of Gα<sup>i</sup> bound

to GDP and inorganic phosphate during GTP hydrolysis (PDB 1GIT).

structure in the Gβ subunit).

Fig. 5. Ribbon representation of Gα subunits in complex with Gβγ or with their effector and regulator proteins, showing the side chains that belong to the protein binding site. The Gα subunit only is shown for simplicity. The amino acids that belong to the protein binding sites of the different Gα structures are shown as space filling. When structurally defined, the ribbon (and side chains) that belong to the N-terminal helix are shown in blue, those that belong to switch 1 in green, to switch 2 in orange, to switch 3 in red, and to the rest of the protein in light grey. Top left: the GDP-bound transducin-Gi chimera showing the interaction surface with Gβγ (1GOT); top center: GTP\*-bound Gα<sup>S</sup> showing the interaction surface with adenylate-cyclase (1CUL); top right: GTP-bound Gα<sup>q</sup> showing the interaction surface with phospholipase-C (3OHM); bottom left:GTP-bound Gα<sup>13</sup> showing the interaction surface with the Rho GEF, p115 (1SHZ); bottom center: GTP-bound transducin-Gi chimera showing the interaction surface with the phosphodiesterase inhibitor subunit, PDEγ (1FQJ) and bottom right, GTP-bound transducin-Gi chimera showing the interaction surface with RGS9 in a complex with PDEγ and RGS9 (1FQK).

Gβγ is also capable of activating certain effectors. Its binding site for Gα overlaps in part the Gβγ-effector and Gβγ- regulator binding sites: the dissociation of GαGTP from Gβγ is essential to allow Gβγ to recognize its effectors (Figure 6).

GTP hydrolysis, rapidly followed by the release of the phosphate ion, modifies the switch regions conformation (Figure 4). The conformation change does not only inhibit the Gαeffector interaction (Figure 5) but also favors Gβγ recognition by GαGDP, thereby also inactivating Gβγ (Figure 6). Agonist-bound receptors interact with both G protein subunits (Figure 3): the formation of the "ternary complex" is an essential step for G protein activation, but the ternary complex Gα subunit is not in the right conformation to activate G protein effectors (Figure 3).

Fig. 6. Ribbon representation of Gβγ showing the interaction surface with Gα or with effectors. Ribbon representation of Gβγ in different crystallized complexes – the Gβ (blue) and Gγ (green) subunits only are shown for simplicity. The amino acids that belong to the Gα recognition surface (PDB ref 2BCG: top), to the phosducin binding site (1GP2: bottom left) or to the kinase, GRK2 binding surface (bottom right : 1OMW) are shown as space filling structures, in yellow.
