Signal Transduction

Table 4.3  G protein receptor kinase family

The G protein receptor kinase family

The phosphorylation of residues in the C-terminal regions of 7TM receptors keeps activation under control. One of the ways in which this occurs is through the phosphorylation of activated receptors by the G protein receptor kinases (GRK 1–7, see Table 4.3). The members of this family have similar structures comprising a kinase domain, an RGS homology (RH) domain and

a more variable C-terminal membrane-targeting region. For instance, the C-terminal residues of the visual GRKs (1 and 7) are hydrophobically modified (farnesylated), while the ubiquitous -adrenergic receptor kinases (GRKs 2 and 3) possess PH domains (see Chapter 24). These bind to the phosphoinositide PI(4,5)P2 and to G protein -subunits, enabling the recruitment of the enzyme from the cytosol.

Receptor phosphorylation, downregulation and pathway switching

The -subunits also serve to present negative feedback signals as demonstrated first through the activation of rhodopsin kinase and later with GRK2/ -adrenergic receptor kinase. Receptors such as muscarinic and adrenergic receptors, phosphorylated at serines and threonines in the C-terminal domain (Figure 4.13), may then recruit adaptor molecules called arrestins. While this is sufficient to block the normal line of communication

through G proteins (receptor desensitization), the bound arrestin can also act as a docking site for a range of signalling molecules, including MAP kinases (Chapter 12), PI 3-kinase (Chapter 18), PKB/Akt (Chapter 18) as well as the soluble protein tyrosine kinase Src. These can initiate new signalling pathways,

98

GTP-Binding Proteins and Signal Transduction

Fig 4.13  The G protein receptor kinase GRK2/ ARK1 binds and subunits at separate sites.Two views of the G i/q·GRK2/G complex are shown as cartoons (top) and as molecular surfaces (bottom). The left-hand structures are side views with the membrane-binding surface at the top. The right-hand structures have been rotated 90° about a horizontal axis. GRK2 possesses a PH domain (orange), an RH domain (pink), and a kinase domain (dark pink). The PH domain binds a -subunit (blue), the RH domain binds the -subunit (mostly green) at sites close to its switch regions (SWI dark blue, SWII red). GDP.AlF4 is shown as spheres in the upper structures (2bcj86).

for example the activation of ERK (see Chapter 12).87 The arrestins can also bind the membrane coat protein clathrin and the associated machinery that allows the receptors to be removed from the plasma membrane by endocytosis.88,89 It is thought that this mechanism is of particular importance in locations, such as sympathetic synapses, where agonist concentrations have a tendency to soar.

As the concentration of the stimulus is raised, two phosphorylating mechanisms, catalysed by protein kinase A (PKA, cAMP-activated protein kinase, see Chapter 9) and by GRK2 are called into action. Since these two kinases have different substrate specificities (consensus sequence selectivities), reacting with different residues in the C-terminal domain of the receptor,

the pattern of phosphorylation is different. It depends on the intensity of stimulation: a mild stimulus inducing PKA activity, or a strong stimulus additionally activating GRK2. The consequence of phosphorylation at the

PKA-reactive sites provides a classic feedback mechanism which acts to disrupt the line of communication with the effector, adenylyl cyclase, and so shuts down the production of cAMP. The phosphorylation due to PKA, which may be maximally activated when less than 10% of the receptors are occupied will also act to down-regulate other receptors having appropriate phosphorylation sites. These will also be prevented from activating cyclase and this is termed heterologous desensitization (other receptors) (Figure 4.14a). Phosphorylation

99

Signal Transduction

Fig 4.14  Heterologous and homologous desensitization by phosphorylation of receptors.(a) Heterologous desensitization. Under mild stimulation the generation of cAMP results in the phosphorylation of all proteins (including receptor molecules) having the PKA substrate consensus motif (-RRSS-). The phosphorylation of receptors occurs regardless of their state of occupation and so the consequence is a generalized down-regulation of all the receptors that regulate cAMP production. (b) Homologous desensitization. Under strong stimulation, the -subunits associated with the receptor act as an anchor for a soluble receptor kinase (GRK2 in the case of2-adrenergic receptors) which phosphorylates only the attached receptor.

may also occur through the action of other second messenger-activated, broad spectrum kinases such as PKC.

By contrast, phosphorylation by receptor-specific kinases affects only activated receptors, for example, GRK2, which is activated by -subunits. This form of inhibition is termed homologous desensitization (same receptor) (Figure 4.14b).

It must be evident that the whole sequence of control, from the first interaction of a hormone with its receptor right through to the generation of second messengers, and then back again to the receptors, is tightly regulated at all stages. Throughout, it remains flexibly sensitive to the needs of the cell.

Receptor mechanisms obviating G proteins

There are a number of apparently anomalous situations in which 7TM receptors are clearly involved, but which appear to cause activation of their target systems without involving G proteins.90 Neither do they involve arrestins or receptor kinases as described in the previous section. Many

of these have been described in neuronal cells and identified initially as changes in the conductance of ion channels. Examples of such non-G- protein-mediated processes are to be found in the activation of non-receptor protein kinases that lead to the activation of the MAP kinase pathway by ligands acting at -adrenergic, acetylcholine (muscarinic), and glutamatergic receptors (for a fuller description of these pathways, see Chapters 12 and 17).

100