35
3
4. G12/13 regulators of actin cytoskeletal remodelling and migration.
5. Transducin, the G-protein that transduces the signal from activated rhodopsins in
the photoreceptor cell. In vertebrates transducin activates a cGMP phosphodiesterase
thus initiating the closing of cGMP-gated ion channels of photoreceptor cells and
membrane hyperpolarization.
Gβ/γ-subunits have a signalling function of their own. They can, for instance, bind to
several types of Ca 2+ -channels (see below) and inactivate them (De Waard et al. 2005).
Moreover, they are known to activate inward rectifying K + -channels (Kir channels or
GIRK channels for those which are activated by G-proteins, see 7 Chap. 4.2) and have
been shown to interact with the MAP kinase pathway, nuclear proteins and the cytoskeleton (Khan et al. 2013) (. Fig. 3.3).
There are two well-known pathogenic modifications of G-proteins. These are catalysed
by ADP-ribosyltransferases that are part of bacterial toxins, including cholera toxin from
Vibrio cholerae and pertussis toxin from Bordetella pertussis. These enzymes catalyse the
transfer of the ADP-ribose element of nicotinamide-adenine dinucleotide to proteins.
Cholera toxin targets G S α-subunits in the intestine and ribosylates an active-site Arg of
the GTP-hydrolase. Thereby, the GTPase activity is blocked, rendering the G-protein constitutively active. As a consequence, cAMP levels in intestinal cells are constantly raising
causing extreme diarrhoea. Pertussis toxin contains an ADP-ribosyltransferase that targets a cysteine residue at position 4 from the C-terminus of G i in the lung epithelium. This
blocks the interaction of the inhibitory G-protein with the receptor and causes an increase
of cAMP in lung epithelial cells resulting in the symptoms of whooping cough. Cholera
and pertussis toxins have provided important tools in G-protein research. For example,
activated cholera toxin together with 32 P-nicotinamide-adenine dinucleotide was used for
the first labelling and purifying of Gα from rabbit liver (Northup et al. 1980).
3.3 G S , G olf and G i Targeting Adenylyl Cyclase
Adenylyl cyclase (AC) is the enzyme that produces cAMP from ATP. There are ten isoforms, expressed in different human tissues. All are regulated by Gα-subunits; these can
be activating (Gsα) or inhibiting (Giα). Some are also activated by the Gβ/γ-dimer that
remains after GTP-bound Gα-subunits are dissociated. Thus, the level of cAMP is regulated via AC in response to extracellular signals, the “first messengers” of intracellular
communication. cAMP is the “second messenger”, produced inside the signal receiving
cells and mediating their responses.
The adenylyl cyclases are integral membrane proteins with 12 hydrophobic transmembrane domains. N- and C-termini are directed into the cell interior. The sequences of the
transmembrane domains are more variable between species than the highly conserved
intracellular loops and the C-terminal sequences. The latter associate with each other to
form a functional unit constituting the catalytic domain of the enzyme. The catalytic
domain of the AC strongly binds to the activator forskolin, a diterpene of the shrub
Plectranthus barbatus that has been used for affinity purification of the AC catalytic
domains from different species (Hatley et al. 2002) (see . Fig. 3.4).
AC activity and the activity of phosphodiesterases (PDEs), which convert cAMP to
AMP and thus antagonize ACs, define the intracellular levels of cAMP. Phosphodiesterases
constitute a large protein family encoded by 21 genes in mammals. Through alternative
splicing the number of known isoforms reaches ca. 50. These are subdivided into separate
3.3 · G S , G olf and G i Targeting Adenylyl Cyclase
3
4. G12/13 regulators of actin cytoskeletal remodelling and migration.
5. Transducin, the G-protein that transduces the signal from activated rhodopsins in
the photoreceptor cell. In vertebrates transducin activates a cGMP phosphodiesterase
thus initiating the closing of cGMP-gated ion channels of photoreceptor cells and
membrane hyperpolarization.
Gβ/γ-subunits have a signalling function of their own. They can, for instance, bind to
several types of Ca 2+ -channels (see below) and inactivate them (De Waard et al. 2005).
Moreover, they are known to activate inward rectifying K + -channels (Kir channels or
GIRK channels for those which are activated by G-proteins, see 7 Chap. 4.2) and have
been shown to interact with the MAP kinase pathway, nuclear proteins and the cytoskeleton (Khan et al. 2013) (. Fig. 3.3).
There are two well-known pathogenic modifications of G-proteins. These are catalysed
by ADP-ribosyltransferases that are part of bacterial toxins, including cholera toxin from
Vibrio cholerae and pertussis toxin from Bordetella pertussis. These enzymes catalyse the
transfer of the ADP-ribose element of nicotinamide-adenine dinucleotide to proteins.
Cholera toxin targets G S α-subunits in the intestine and ribosylates an active-site Arg of
the GTP-hydrolase. Thereby, the GTPase activity is blocked, rendering the G-protein constitutively active. As a consequence, cAMP levels in intestinal cells are constantly raising
causing extreme diarrhoea. Pertussis toxin contains an ADP-ribosyltransferase that targets a cysteine residue at position 4 from the C-terminus of G i in the lung epithelium. This
blocks the interaction of the inhibitory G-protein with the receptor and causes an increase
of cAMP in lung epithelial cells resulting in the symptoms of whooping cough. Cholera
and pertussis toxins have provided important tools in G-protein research. For example,
activated cholera toxin together with 32 P-nicotinamide-adenine dinucleotide was used for
the first labelling and purifying of Gα from rabbit liver (Northup et al. 1980).
3.3 G S , G olf and G i Targeting Adenylyl Cyclase
Adenylyl cyclase (AC) is the enzyme that produces cAMP from ATP. There are ten isoforms, expressed in different human tissues. All are regulated by Gα-subunits; these can
be activating (Gsα) or inhibiting (Giα). Some are also activated by the Gβ/γ-dimer that
remains after GTP-bound Gα-subunits are dissociated. Thus, the level of cAMP is regulated via AC in response to extracellular signals, the “first messengers” of intracellular
communication. cAMP is the “second messenger”, produced inside the signal receiving
cells and mediating their responses.
The adenylyl cyclases are integral membrane proteins with 12 hydrophobic transmembrane domains. N- and C-termini are directed into the cell interior. The sequences of the
transmembrane domains are more variable between species than the highly conserved
intracellular loops and the C-terminal sequences. The latter associate with each other to
form a functional unit constituting the catalytic domain of the enzyme. The catalytic
domain of the AC strongly binds to the activator forskolin, a diterpene of the shrub
Plectranthus barbatus that has been used for affinity purification of the AC catalytic
domains from different species (Hatley et al. 2002) (see . Fig. 3.4).
AC activity and the activity of phosphodiesterases (PDEs), which convert cAMP to
AMP and thus antagonize ACs, define the intracellular levels of cAMP. Phosphodiesterases
constitute a large protein family encoded by 21 genes in mammals. Through alternative
splicing the number of known isoforms reaches ca. 50. These are subdivided into separate
3.3 · G S , G olf and G i Targeting Adenylyl Cyclase