**8.1 Activation of several G protein subtypes by the same receptors**

Each cell expresses several G proteins, belonging or not to the same family: all these G proteins will compete for recognition of each activated GPCR. Most Gi-coupled receptors activate several Gi isoforms with variable efficiency; some Gi and GS coupled receptors activate in addition Gq/11 G proteins - less efficiently, or only at much higher agonist concentrations... Does this reflect a lower (but measurable) affinity of the non-cognate G protein, or less efficient activation?

GPCRs catalyze G protein activation: they should be considered like honorary enzymes. If several substrates compete for transformation by the same enzyme, the proportion of substrates transformed by the enzyme per minute, at steady state, is proportional to their relative substrate concentration over specificity constant ratios, [S]/KS :

$$\frac{\upsilon^{\wedge}}{\upsilon^{\wedge}} = \frac{\left[A\right] / \mathcal{K}\_{\text{s}}^{\wedge}}{\left[B\right] / \mathcal{K}\_{\text{s}}^{\wedge}}\tag{1}$$

(where A and B represent the two substrates (G proteins), respectively, and *<sup>A</sup> KS* and *<sup>B</sup> KS* are their respective specificity constants : *cat S M <sup>k</sup> <sup>K</sup> <sup>K</sup>* ).

The equation is extremely similar to the equation describing the competition of several ligands for the same receptor: the proportion of receptor occupied by each ligand ([RA] and [RB]) is proportional to their relative ligand concentration over dissociation constant ratios:

$$\frac{\left[RA\right]}{\left[RB\right]} = \frac{\left[A\right]\left/K\_{\rm D}^{\rm A}}{\left[B\right]\left/K\_{\rm D}^{\rm B}} \text{ (2)}$$

The meaning of "KD" and "KS" is however very different: the dissociation constant, KD = 1/Kaffinity, is a concentration. It measures the ligand concentration necessary to occupy, at equilibrium and in the absence of competitors, 50% of the receptors. The specificity constant KS, in contrast is a bimolecular reaction rate constant and measured in M-1sec-1. It measures the rate of formation of the "productive complex", ES† in the absence of alternative substrates of inhibitors.

Multiple G protein signaling has more often been observed in transfected systems, where it depends on the receptor expression level (for review: (Hermans, 2003)). Transiently expressed α2 adrenergic receptors inhibit adenylate cyclase at low agonist concentrations but activate the enzyme at high agonist concentrations (Fraser et al., 1989). Adenylate cyclase inhibition but not activation is prevented by Gi protein inactivation by pertussis toxin (Fraser et al., 1989): these results indicate that α2 adrenergic receptors are capable of activating both Gi and GS. The equations above predict that the relative activation rate of "Gi" and "GS" is proportional to their relative concentrations. Activation of GS by α2 adrenergic receptors is observed only at very high agonist concentrations: this suggests that, at very high agonist concentrations, Gi becomes unable to compete for receptor activation: in contrast with Gi-GDP, the activated Gi-GTP complex is probably unable to recognize agonist-bound α2 adrenergic receptors (Waelbroeck, 2001).

A few GPCRs are capable of activating several G proteins in physiological settings: the G protein specificity is not always "absolute". Although this is unusual in Family A, some G protein coupled receptors can be expressed as related isoforms due to alternative splicing of RNA expressed from a single gene or to RNA editing: this may lead to receptor isoforms with different abilities to activate G proteins (Hermans, 2003; Bresson-Bepoldin *et al.*, 1998). Alternatively, post-translational modifications such as phosphorylation of the receptor may alter its G protein specificity: β2-adrenergic receptors activate GS proteins, leading to adenylate cyclase and protein kinase A stimulation, then – after phosphorylation by protein kinase A – activate Gi proteins (Zamah *et al.*, 2002). The TSH receptor is able to activate G proteins from all four families (Allgeier *et al.*, 1997; Laugwitz *et al.*, 1996). Its binding properties are compatible with the hypothesis that it forms a stable dimer, and that occupancy of the dimer by one TSH molecule decreases the affinity of the second binding

 *A A*

*v A K*

(where A and B represent the two substrates (G proteins), respectively, and *<sup>A</sup> KS* and *<sup>B</sup> KS* are

*M*

The equation is extremely similar to the equation describing the competition of several ligands for the same receptor: the proportion of receptor occupied by each ligand ([RA] and [RB]) is proportional to their relative ligand concentration over dissociation constant ratios:

> 

The meaning of "KD" and "KS" is however very different: the dissociation constant, KD = 1/Kaffinity, is a concentration. It measures the ligand concentration necessary to occupy, at equilibrium and in the absence of competitors, 50% of the receptors. The specificity constant KS, in contrast is a bimolecular reaction rate constant and measured in M-1sec-1. It measures the rate of formation of the "productive complex", ES† in the absence of alternative

Multiple G protein signaling has more often been observed in transfected systems, where it depends on the receptor expression level (for review: (Hermans, 2003)). Transiently expressed α2 adrenergic receptors inhibit adenylate cyclase at low agonist concentrations but activate the enzyme at high agonist concentrations (Fraser et al., 1989). Adenylate cyclase inhibition but not activation is prevented by Gi protein inactivation by pertussis toxin (Fraser et al., 1989): these results indicate that α2 adrenergic receptors are capable of activating both Gi and GS. The equations above predict that the relative activation rate of "Gi" and "GS" is proportional to their relative concentrations. Activation of GS by α2 adrenergic receptors is observed only at very high agonist concentrations: this suggests that, at very high agonist concentrations, Gi becomes unable to compete for receptor activation: in contrast with Gi-GDP, the activated Gi-GTP complex is probably unable to recognize

A few GPCRs are capable of activating several G proteins in physiological settings: the G protein specificity is not always "absolute". Although this is unusual in Family A, some G protein coupled receptors can be expressed as related isoforms due to alternative splicing of RNA expressed from a single gene or to RNA editing: this may lead to receptor isoforms with different abilities to activate G proteins (Hermans, 2003; Bresson-Bepoldin *et al.*, 1998). Alternatively, post-translational modifications such as phosphorylation of the receptor may alter its G protein specificity: β2-adrenergic receptors activate GS proteins, leading to adenylate cyclase and protein kinase A stimulation, then – after phosphorylation by protein kinase A – activate Gi proteins (Zamah *et al.*, 2002). The TSH receptor is able to activate G proteins from all four families (Allgeier *et al.*, 1997; Laugwitz *et al.*, 1996). Its binding properties are compatible with the hypothesis that it forms a stable dimer, and that occupancy of the dimer by one TSH molecule decreases the affinity of the second binding

*RA A K RB B K* (2)

*A D B D*

*S*

 

*<sup>k</sup> <sup>K</sup> <sup>K</sup>* ).

their respective specificity constants : *cat*

agonist-bound α2 adrenergic receptors (Waelbroeck, 2001).

substrates of inhibitors.

*B B*

*S*

*v BK* (1)

*S*

site ("negative cooperativity" (Urizar *et al.*, 2005)). While signaling through GS is induced at very low TSH concentrations, low affinity occupancy of two binding sites per dimer appears to be necessary to drive receptor activation of Gi (Allen *et al.*, 2011).
