-subunits. An important end result of this switching is the involvement of Ras leading to activation of ERK (extracellular regulated kinase) and the generation of nuclear signals (Figure 9.5). Further details will be given in Chapter 12.

This line of communication is interrupted in cells that have been treated with pertussis toxin, a characteristic of the involvement of Gi proteins, not Gs. It can also be prevented by over-expression of a peptide containing a PH domain, derived from the C-terminus of the receptor kinase GRK2 (GRK2/ ARK: see page 98) which binds to -subunits. All this effectively places cAMP and PKA upstream of the receptor, conditioning it to communicate with Gi and then Ras, leading to the activation of ERK (Chapter 12).

Actions of cAMP not mediated by PKA

Regulation of ion channels by cyclic nucleotides

Important among the actions of cAMP that are not mediated through PKA is the regulation of a gated cation channel, a component of the signalling mechanism in olfactory cells27–30 (see page 179).

Epac, a guanine nucleotide exchange factor directly activated by cAMP

Rap1 is a monomeric GTPase involved in a number of cellular processes that include the activation of platelets, cell proliferation, differentiation, and

FIG 9.5  All change trains. Down-regulated receptors are not necessarily down-regulated, merely looking the other way.

Phosphorylation of the 2-adrenergic receptor by PKA diverts its attention from Gs to Gi. The resulting activation of -subunits initiates a pathway of reactions leading to the activation of ERK (extracellular signal regulated kinase).

Adapted from Daaka et al.26

The cAMP gated K channel is composed of four similar protein

chains that are assembled together so that the transmembrane domains (generally spanning

the membrane five or six times) combine to form a central pore. In the voltage-gated Na and Ca2 channels the subunits are linked as a single protein chain.

FIG 9.6  Activation of Rap1 by Epac, a guanine nucleotide exchange factor activated by cAMP.

Rap1 is a monomeric GTPase that is confined to organelle membranes and that counteracts the oncogenic effects of Ras. Its activation is an example of a process mediated by cAMP without the participation of PKA.

morphogenesis.31 Although initially identified as a K-Ras-related protein,32 Rap1 does not transform but rather antagonizes the action of Ras33 (Figure 9.6). Unlike Ras, Rap1 is confined to organelle membranes. This may explain how, despite the two proteins sharing some downstream effectors, their effects can be antagonistic. Depending on the cell type, several ligands are able to activate Rap1, through interaction with their receptors and following the generation of second messengers such as Ca2 , diacylglycerol (DAG), and cAMP. It is important to note, however, that this works equally well in cells that express a mutant form of PKA that has a low affinity for cAMP.

The activation of Rap1 is mediated through the direct action of cAMP with a guanine nucleotide exchange factor (Epac, exchange protein directly activated by cAMP).34 This was identified by screening sequence databases for those proteins that have homology with GEFs for Ras and Rap1 and also

binding sites for cAMP. The nucleotide binding site is similar to those present in the regulatory subunits of PKA and the cyclic nucleotide-regulated ion channels. Using purified Epac it was shown that cAMP is required to initiate the release of GDP from Rap1. The interaction of a mutant form of Epac, lacking the cyclic nucleotide binding site, is constitutively active. It can release GDP from Rap1 in the absence of cAMP.

As with to other monomeric GTPases, the return to the GDP-bound state is promoted by interaction with a GTPase activating protein. The specific

Rap1GAP is itself regulated by the heterotrimer Go in its resting state.35 Thus,o.GDP can sequester Rap1GAP and in this way regulate the various activities of Rap1. We return to Rap1 and integrin activation in Chapter 16 (page 498).