Knockdown of AKAP Expression Using RNA Approaches

AKAP350 targets PKA and a variety of interacting proteins to the Golgi apparatus in epithelial cells. Larocca et al. utilized RNA directed against AKAP350, which reduced the expression level to 20% in HeLa cells and disperses the Golgi apparatus, indicating that the AKAP is important for the maintenance of the Golgi.

The human AKAP79 and its rat ortholog AKAP150 bind PKA, PKC and calcineurin. Knockdown of AKAP79 in HEK293-B2 cells, stably expressing the P2-adrenoceptor, revealed that AKAP79-bound PKA phosphorylates the receptor and thereby induces switching of the receptor G protein coupling from Gs to Gi (Lynch et al. 2005).

The use of RNA also revealed that AKAP79/AKAP150 coordinate the regulation of AMPA receptors and M-type K+ channels (Hoshi et al. 2005; Hoshi and Scott 2006). Endogenous AKAP79 in human-derived cells and AKAP150 in rat-derived cells were knocked down. Their expression was reconstituted with an ortholog version lacking a particular binding domain. For example, the lack of AKAP79 was rescued by an AKAP150 version lacking the PKA-binding domain. This work showed that in hippocampal neurons, AKAP150 positions PKA and calcineurin to modulate AMPA channels and maintains PKC inactive. In superior ganglial neurons, AKAP150 facilitates PKC phophorylation of M channels, while keeping PKA and calcineurin inactive. The difference in targeting is due to the interaction of AKAP150 with the scaffolding protein SAP97, which occurs in hippocampal neurons, but not in superior ganglial neurons.

The expression and thus RhoGEF activity of AKAP-Lbc and the expression of mAKAP are upregulated in cardiac myocyte hypertrophy. The AKAP-Lbc RhoGEF activity upregulation is due to constitutive ^-adrenoceptor signalling (see above). Knockdown of either AKAP-Lbc or mAKAP prevents the hypertrophy (Pare et al. 2005; Appert-Collin et al. 2007).

AKAP185 directly interacts with phospholamban (PLN) in cardiac myocytes. Knockdown of AKAP185 decreases the velocity of Ca2+ reuptake from the cytosol into the sarcoplasmic reticulum (SR). Therefore, AKAP185 participates in controlling the relaxation of the heart muscle (Lygren et al. 2007).

Willoughby et al. (2006) compared cAMP dynamics in microdomains beneath the plasma membrane with global cAMP dynamics in HEK293 cells. RNAi directed against human gravin (also termed AKAP12 or AKAP250) reduces gravin expression by 80% and limits hydrolysis of subplasmalemmal cAMP, as gravin no longer tethers PDE4D to the plasma membrane. Rescue of gravin expression with the mouse ortholog Src-suppressed C kinase substrate (SSeCKS) of gravin reverses the effects of gravin knockdown (Willoughby et al. 2006).

Combined knockdown of MAP2 or tau in MAP1B-deficient mouse hippocam-pal neurons with antisense oligonucleotides arrests the cells in early stages of neuronal polarity development (compare MAP2 knockout mice, see Sect. 2.1; Gonzalez-Billault et al. 2002).

AKAP97 (radial spoke protein 3, RSP3) is a flagellar protein of Trypanosoma brucei. Its knockdown leads to immotility due to defective flagellar beat, loss of radial spokes and defects in cytokinesis (Ralston et al. 2006). In nasopharyngeal carcinoma 5-8 F cells, RNA directed against ezrin reduces the invasiveness of the cells (Peng et al. 2007).

Similar to the knockout approaches described above (Sect. 2.1), knockdown strategies have highlighted crucial roles of AKAPs in a variety of cellular functions that cause disease if they are dysregulated. However, like the knockout strategies, knockdown approaches do not define the roles of AKAP-PKA or other direct AKAP-mediated protein-protein interactions in a cellular process. In order to define the function of particular protein interactions, agents such as peptides or small molecules for their disruption are needed.

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