A-subunit, G protein

Pertussis toxin is produced by the bacterium Bordetella pertussis. It covalently modifies G-proteins of the G/Go family (transfer of a ADP-ribose moiety of NAD onto G-protein a-subunits). ADP-ribosylated G-proteins are arrested in their inactive state and, as a consequence, functionally uncoupled from their respective effectors. Examples for pertussis toxin-sensitive cellular responses include the hormonal inhibition of adenylyl cyclases, stimulation ofK+ channels, inhibition of Ca2+ channels and stimulation ofthe cGMP-phosphodiesterase in retinal rods. 

The inner face of the activated receptor binds to the C-terminus of the G-protein a subunit (see Figure 7.3). Inner loop 3 (ic3) between transmembrane helices 5 and 6 of the receptor plays a critical role in this interaction. Note, however, that although the a subunit bears the primary binding site for the receptor, attachment of the py-dnner to the a subunit is essential for this interaction to occur. 

Binding of GTP promotes a disordering of the carboxyl- and amino-termini of the G-protein a subunit, with two parallel consequences the GTP-bound a subunit dissociates... 

The terminal (y) phosphate of GTP is hydrolyzed by the GTPase activity of the G-protein a subunit, leaving GDP bound instead. This reverses the conformational change in step 5 and allows the a subunit to dissociate from the effector and reassociate with the py subunit. The reassociation will also reverse Py-effector interaction because Ga-GDP effectively competes with the effector for Py-binding. Though of fairly high affinity (e.g.,... 

FIGURE 7.7 C-terminal residues of G-protein a subunits. The cysteine ADP-ribosylated by Pertussis toxin (PTx) is boxed. 

The first G-protein a subunit to be identified was Gs. The a subunit of Gs (as) is responsible for stimulating adenylate cyclase (hence, the subscript s ) and is ADP-ribosylated and activated by CTx. Gs has at least four molecular variants. Some evidence exists that as can also enhance the activity of cardiac L-type Ca2+ channels, independently of their phosphorylation by cAMP-stimu-lated protein kinase A. Golf is a cyclase-stimulating homolog in the olfactory epithelium, activated by the large family of olfactory receptors. 

In addition to G protein a subunits, / subunits of G proteins also associate with 8 receptors and may be involved in 8 receptor signaling. Antisera directed against... 

G proteins can be modified by ADP-ribosylation catalyzed by certain bacterial toxins. Among the tools that facilitated the discovery and characterization of G proteins were the bacterial toxins cholera and pertussis, which were known to influence adenylyl cyclase activity. Subsequently, it was shown that the actions of these toxins are achieved by their ability to catalyze the addition of an ADP-ribose group donated from nicotinamide adenine dinucleotide (NAD) to specific amino acid residues in certain heterotrimeric G protein a subunits [ 1 ]. 

In contrast, pertussis toxin catalyzes the ADP-ribosyl-ation of a specific cysteine residue in Gai) G(m and Gal [1]. Only a subunits bound to their Py subunits can undergo this modification. Pertussis-toxin-mediated ADP-ribosylation inactivates these a subunits such that they cannot exchange GTP for GDP in response to receptor activation (Fig. 19-1B). By this mechanism, pertussis toxin blocks the ability of neurotransmitters to inhibit adenylyl cyclase or to influence the gating of K+ and Ca2+ channels in target neurons. However, since G is not a substrate for pertussis toxin, the toxin may not be able to block neurotransmitter-mediated inhibition of adenylyl cyclase in all cases. The Gq and Gn 16 types of G protein a subunit are not known to undergo ADP-ribosylation. 

Protein present in Fabl, YOTB, Vacl, and EEA1 G protein a subunit... 

The G proteins with which G protein-coupled receptors interact are heterotrimeric complexes composed of a, P, and y subunits. In its inactive state, the G-protein a subunit is bound to GDP and associates with a Py dimer. Upon receptor activation, the a subunit opens its guanine nucleotide binding site and releases its bound GDP, allowing GTP, which is present in higher concentrations in the cell, to bind in its place. The GTP-bound a subunit has a lower affinity for the Py dimer and dissociates from it, freeing both the a sub-... 

Recently, it was discovered that G-protein activity is controlled by another class of proteins. These proteins, termed RGS proteins (for regulators of G-protein signaling), enhance the rate of GTP hydrolysis. This activity reduces the amount of time the G-protein a subunit spends in its active GTP-bound conformation, in effect limiting the response to receptor activation. 

Fig. (9). A) Schematical representation of the activation of intact adenylate cyclase (AC) b forskolin and Gsa (GTP- bound stimulatory G protein a subunit). Mammalian ACs consists o 12 transmembrane helices and two cytoplasmic catalytic domains (referred to as Q and C2 represented as lightly shaded and black respectively), (a) Hypothetical basal state, (b) Th suggested forskolin-activated state, (c) Forskolin and GSa-activated state.

The adenylate cyclase pathway of hormone receptor action. When the receptor is unoccupied, the G protein a subunit has GDP bound, and it is complexed with the subunits in this form it cannot activate adenylate cyclase. Binding of hormone activates the receptor, which leads to replacement of GDP by GTP, and the activation subunit then interacts productively with adenylate cyclase to stimulate the synthesis of cAMP. The intrinsic GTPase activity of the a subunit leads to... 

Berman, D. M., Wilkie, T. M., and Gilman, A. G. (1996b). GAIP and RGS4 are GTPase-activating proteins (GAPs) for the G subfamily of G protein a subunits. Cell 86, 445-452. 

Zelent, B., Veklich, Y., Murray, J., Parkes, J. H., Gibson, S., and Liebman, P. A. (2001). Rapid irreversible G protein a subunit misfolding due to intramolecular kinetic botdeneck that precedes Mg2+ lock after GTP/GDP exchange. Biochemistry 40, 9647-9656. 

Extreme C terminus of G protein a-subunits contains a site that discriminates between Gj-coupled metabotropic glutamate receptors. /. Biol. Chem. 273, 25765-25769. 

Heydorn, A., Ward, R. J., Jorgensen, R., Rosenkilde, M. M., Frimurer, T. M., Milligan, G., and Kostenis, E. (2004). Identification of a novel site within G protein a subunits important for specificity of receptor-G protein interaction. Mol. Pharmacol. 66, 250-259. 

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