Disruption of Akappka Interactions by Genetically Encoded Peptides

In order to study the function of PKA anchoring in cell and animal models, disruptor peptides have been genetically encoded for expression. Generally, this approach overcomes limitations of peptides, such as their short half-life if applied to animals parenterally. In addition, this technique allows for introduction of peptides into cells and possibly animals that are not or are only limitedly accessible to chemical synthesis. Furthermore, encoding peptides genetically permits labelling with chromophores, such as green fluorescent protein (GFP), permitting tracking of the peptides in living cells. For example, a GFP-AKAPIS fusion protein transiently expressed in HEK293 cells displaces PKA from the Golgi and was utilized to study co-localisation with PKA RII subunits (Alto et al. 2003). Similarly, the peptide RIAD was fused to GFP. Analysis of HEK293 cells expressing the fusion protein revealed binding of RIAD to PKA regulatory RI subunits and confirmed the involvement of PKA type I in T cell receptor signalling and progesterone production (see above).

Bond and co-workers introduced Ht31 peptides by adenoviral transfer into primary neonatal cardiac myocytes and observed an increase in contractility (see Sect. 3; Fink et al. 2001).

In order to investigate the influence of AKAP-PKA interactions on hippocampal synaptic plasticity and spatial memory, transgenic mice conditionally expressing the peptide Ht31 in forebrain neurons were generated (Nie et al. 2007). Analysis of the transgenes shows that at hippocampal Schaffer collateral CA3-CA1 synapses,

Table 2 Examples of functional consequences of disruption of AKAP-dependent protein-protein interactions with disruptor peptides (see Table 1)

Peptide

Protein complex

disruptor

Cell type

comprising

Functional consequence

Ht31

Mouse oocytes

PKA, AKAP?,

Stimulation of oocyte

maturation

maturationa

promoting factor

S-Ht31

Renal inner medul

AKAP18S, AKAP?,

Inhibition of aquaporin-2

lary collecting duct

PKA

redistributionb

(IMCD) cells

AKAP188-wt

Rat neonatal cardiac

PKA, AKAP18a,

Reduction of L-type Ca2+

myocytes

L-type Ca2+ channel

channel currentsc

AKAPjs

HEK293 cells

PKA, AKAP79, AMPA

Reduction of ectopically

channel (GluRl

expressed GluR1

receptor subunits)

receptor currentsd

RIAD-Arg11

T cells

PKA Type I, Ezrin

Reduction of Lck

phosphorylation (acti-

vation of cAMP inhib-

ited T cell signalling)e

RIAD-Arg11

Mouse Y1

PKA Type I, PBR

Reduction of progesterone

adrenocortical cells

productione

superAKAP-IS

Hippocampal neurons

PKA, AKAP150, GluRl

Reduction of

receptor subunits

AMPA-receptor

of AMPA channel,

currentsf

PP2B

AKAP15-LZ

Mouse skeletal mus

AKAP18a, PKA, L-

Reduction of

cle cells (MM14,

type Ca2+ channel

voltage-dependent

DZ1A)

(Cav1.1)

potentiation of Ca2+

channelsg

Arg9-11-PLN

Rat neonatal and adult

PKA, AKAP188, PLN,

Reduction of Ca2+ re-

cardiac myocytes

SERCA2

uptake into the SRh

Listed are the peptides utilized (peptide disruptors), components comprising the macromolecular complexes targeted by the peptides and the cell types in which the peptides elicited the indicated functional consequences. The peptides Ht31, S-Ht31, AKAP18S-wt, AKAP18S-L304T, AKAP18S-L314E, RIAD-Arg11 and SuperAKAP-IS disrupt AKAP-PKA interactions. The peptides AKAP15-LZ and Arg9-11-PLN disrupt AKAP18a-L type Ca2+ channel and AKAP18S-PLN interactions, respectively. ?, unidentified AKAP; AMPA, a-amino-3-hydroxy-5-methyl-4-isoxa-zolepropionic acid; PAP7, PBR-associated protein; PBR, peripheral-type benzodiazepine receptor; PLN, phospholamban; PP2B, protein phosphatase 2B/calcineurin A1; SERCA2, sarcoplasmic reticulum Ca2+-ATPase 2; SR, sarcoplasmic reticulum a Newhall et al. (2006)

b Klussmann et al. (1999), Henn et al. (2004) c Hundsrucker et al. (2006) d Alto et al. (2003) e Carlson et al. (2006) f Gold et al. (2006) g Hulme et al. (2002) h Lygren et al. (2007)

long-term potentiation (LTP) requires presynaptically anchored PKA if the neurons are stimulated by a theta-burst and postsynaptically anchored PKA if multiple high-frequency trains are used for stimulation. In addition, the data show that a pool of anchored PKA in CA3 neurons is required for spatial memory storage.

A role of compartmentalized cAMP signalling in memory formation of Drosophila was revealed by utilizing a peptide (eCOPR2) mimicking the AKAP-binding domain in the N terminus of Drosophila RII subunits (Schwaerzel et al. 2007). The peptide blocks interactions of RII subunits with AKAPs. It was expressed ubiquitously throughout Drosophila or in defined parts of the Drosophila nervous system. With this system, AKAP-bound pools of PKA were detected in distinct regions of the nervous system, and AKAP-dependent functions during aversive memory formation were elucidated.

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