cAMP effector proteins

As described, cAMP is produced by membrane bound ACs which are activated via Gs protein-mediated signalling. cAMP is known to act through interaction with and activation of four distinct classes of effector proteins: protein kinase A (PKA), exchange protein directly activated by cAMP (Epac), hyperpolarisation- activated cyclic nucleotide-gated cation (HCN) channels and Popeye domain containing (POPDC) proteins (see Figure 1.3).

Figure 1.3 Signalling pathways of the four cAMP effector proteins and their main functions in the heart. Norepinephrine (NE) is secreted by sympathetic neurons and can bind to the β- adrenergic receptor embedded in the membrane of the cell. Binding leads to Gs activation and subsequent activation of Adenylyl Cyclase’s (AC) generating cAMP. AC are inhibited through acetylcholine ligand binding to the muscarinic acetylcholine receptor. Upon receptor activation, Gi can be activated leading to the inhibition of AC decreasing cAMP production. Four main cAMP effector proteins sense these changes in cAMP levels; protein kinase A (PKA), exchange factor directly activated by cAMP (EPAC), hyperpolarization activated cyclic nucleotide gated (HCN) channel, the Popeye domain containing (POPDC) protein. PKA and EPAC are usually found bound to the same A-kinase anchoring protein (AKAP), whereas, POPDC1 and CNGCs are membrane bound. Listed below each effector protein are the main cardiac processes each is involved in. (Figure adapted from (Brand, 2018))

1.2.3.1 PKA

PKA was one of the first protein kinases to be discovered (Walsh et al., 1968). Interestingly, unlike most other eukaryotic protein kinases, PKA exists as a tetrameric enzyme consisting of two regulatory (R) subunits and two catalytic (C) subunits (Kemp et al., 1975). The C subunit of PKA can be phosphorylated at Threonine 197 (T197) which resides within the activation loop (Cheng et al., 1998, Cauthron et al., 1998). The C subunit is regulated by the interaction with the inhibitory R subunit. The R subunit binds to cAMP driving a conformational

change that leads to their dissociation from the C subunits. This dissociation allows for activation of the catalytic activity of the enzyme which can affect a range of diverse cellular processes by phosphorylating numerous cytoplasmic and nuclear proteins (Taylor et al., 1990). There are two known classes of PKA

designated as PKA (I) and PKA (II) due to the differences that are found within R subunits, RI and RII respectively, that can interact with the identical C subunit (Taylor et al., 1990). Although both RI and RII contain tandem and highly

conserved cAMP-binding domains (CBD) they differ in the N-terminal proteolytic hinge that allows their binding to the recognition site in the C subunit. In RII subunits there is a serine residue that can be autophosphorylated by the C subunit while the RI subunit possesses a pseudo phosphorylation site (Rosen and Erlichman, 1975).

PKA is a member of the family of serine/ threonine kinases that uses the active site to catalyse the transfer of a phosphate group from ATP to a threonine or serine residue on the target protein. Target threonine/serine residues are usually found integrated into a R-R-x-S/T-θ motif, where θ represents a

hydrophobic amino acid and x represents any amino acid (Skalhegg and Tasken, 2000). As mentioned, this phosphorylation drives the activation of numerous cellular processes. For example, in the heart PKA is known to be an influential cAMP effector protein involved in excitation-contraction coupling (described in section 1.1) via direct phosphorylation and activation of L-type calcium channels (LTCC), troponin I (TnI), myosin binding protein C (MyBP-C), phospholamban (PB), and potassium channels (Wolff et al., 1996, Baryshnikova et al., 2008, Barefield and Sadayappan, 2010, Kurokawa et al., 2004).

1.2.3.2 Epac

A more recently identified cAMP effector protein is exchange protein directly activated by cAMP (Epac). It was found to have a cAMP binding domain

homologous to that of PKA R subunits (de Rooij et al., 1998, Kawasaki et al., 1998). Epac is known to bind cAMP with high affinity and functions to activate the Ras family small GTPases Rap1 and Rap2 (Cheng et al., 2008). There are two isoforms of Epac, Epac1 and Epac2, which are the products of two independent genes in mammals and share extensive sequence homology. Both Epac1 and Epac2 possess distinctly different N-terminal regions whereas they share an

evolutionary conserved catalytic domain. Each isoform has a unique expression pattern with Epac1 being ubiquitously spread while Epac2 has a more restricted pattern of tissue expression (de Rooij et al., 1998, Kawasaki et al., 1998). These proteins are involved in many crucial cellular processes such as cell adhesion and migration through activation of integrins (Rangarajan et al., 2003, Han et al., 2006). Many of the processes that are driven by EPAC are also regulated by the activity of PKA. For example, PKA is also known to phosphorylate Rap1 at a site in the C-terminal however PKA phosphorylation is not necessary for the cAMP- dependent activation of Rap1 (de Rooij et al., 1998).

1.2.3.3 CNGC

Ions channels that are directly activated by cyclic nucleotides were first

discovered during the hunt for the second messenger that is crucial in mediating the response of retinal photoreceptors (Haynes and Yau, 1985). These channels are predominantly activated by cyclic guanosine monophosphate (cGMP) (Liu et al., 1994). CNGCs belong to a superfamily of heterogeneous ion channels that are linked by the fact that they possess a common transmembrane and pore structure. Within the C-terminus there exists a binding site for 3’5’-cyclic nucleotide monophosphates (cNMPs) (Kaupp and Seifert, 2002).

Each CNGC is composed of four subunits which each contain six transmembrane domains followed by the C-terminus, which housing the cyclic nucleotide binding domain (CBD) (Kaupp and Seifert, 2002, Matulef and Zagotta, 2003). Binding of cNMPs to the CNCG results in the opening of the channel and subsequently the influx of cations into the cell.

The hyperpolarisation and cyclic nucleotide-gated channel (HCNGC) is of

particular importance in the maintenance of the “funny” pacemaker current (If)

in the heart (Ludwig et al., 1998, Santoro et al., 1998). There are four isoforms of HCN (HCN1-4), which are all known to be differentially expressed within the heart (Scicchitano et al., 2012). Channels are modulated by the direct action of cAMP binding which induces conformational changes that allow for the opening of the channel during hyperpolarisation of the cardiac membrane (DiFrancesco and Tortora, 1991). In addition, the binding of cAMP allows for the lowering of the threshold potential of the channel opening (DeBerg et al., 2016). During the

opening, the channels become permeable to Na+ _{and K}+ _{ions that are close to the}

resting membrane potential of the cell producing the If current (Benarroch,

2013). Generation of the If current is responsible for the initial depolarisation

of the cardiac action potential (DiFrancesco and Borer, 2007).

1.2.3.4 POPDC

The newest class of cAMP effectors to be identified are the Popeye domain containing (POPDC) gene family (Andrée et al., 2000, Reese et al., 1999).