The Role of PDZDomain Containing Scaffolds

Recent studies have exposed PAR C-terminal domain interactions with PDZ-domain-containing scaffolds as a mechanism that controls PAR targeting in cells (Fig. 1). The extreme C-termini of P1 and P2 ARs conform to type-1 PDZ-domain-binding sequences (S/T-X-^, where indicates a hydrophobic amino acid and "X" indicates any amino acid); these PAR subtypes interact with different sets of PDZ-domain-containing proteins. The PjAR-ESKY motif associates with structurally

Fig. 1 PARs complex with three types of scaffolds. (a) P1 and P2ARs interact with PDZ-domain-containing scaffolding proteins. SAP97 links PtARs to the AKAP79-PKA complex, whereas NHERF Links P2ARs to the Na+-H+ exchanger 3 (NHE3) and the actin-associated protein ezrin. An interaction between ezrin and a membrane-proximal portion of the alb-AR C-tail is required for alb-AR recycling to the plasma membrane. (b) The initial PAR signal via the cAMP/PKA pathway is followed by a subsequent activation of the ERK cascade in many cell types. Two mechanisms have been identified: PKA phosphorylation switches PAR coupling from Gs to Gi. P-arrestin docks to GRK-phosphorylated PARs (and other GPCRs) and functions to interdict signaling by G proteins, to recruit PDE4, and to assemble signaling proteins that regulate growth/ apoptosis signaling pathways. (c) An association between the PAR C-terminus and AKAP79/150 or gravin contributes to the spatial and temporal control of signaling through the PKA pathway (See text for details).

Fig. 1 PARs complex with three types of scaffolds. (a) P1 and P2ARs interact with PDZ-domain-containing scaffolding proteins. SAP97 links PtARs to the AKAP79-PKA complex, whereas NHERF Links P2ARs to the Na+-H+ exchanger 3 (NHE3) and the actin-associated protein ezrin. An interaction between ezrin and a membrane-proximal portion of the alb-AR C-tail is required for alb-AR recycling to the plasma membrane. (b) The initial PAR signal via the cAMP/PKA pathway is followed by a subsequent activation of the ERK cascade in many cell types. Two mechanisms have been identified: PKA phosphorylation switches PAR coupling from Gs to Gi. P-arrestin docks to GRK-phosphorylated PARs (and other GPCRs) and functions to interdict signaling by G proteins, to recruit PDE4, and to assemble signaling proteins that regulate growth/ apoptosis signaling pathways. (c) An association between the PAR C-terminus and AKAP79/150 or gravin contributes to the spatial and temporal control of signaling through the PKA pathway (See text for details).

related membrane-associated guanylate kinase-like (MAGUK) PDZ family proteins, including postsynaptic density protein 95 (PSD-95), membrane-associated guanylate kinase-like protein inverted-2 (MAGI-2), MAGI-3, and SAP97, as well as GIPC (GAIP-interacting protein, carboxyl terminus), and cystic fibrosis transmembrane conductance regulator-associated ligand (CAL, a protein primarily involved in vesicular transport in the Golgi) (Hu et al. 2000; Xu et al. 2001; Hu et al. 2003; He et al. 2004, 2006; Gardner et al. 2007). PSD-95 is reported to inhibit P1AR internalization, whereas MAGI-2 exerts a diametrically opposite effect to promote P1AR internalization, indicating that individual MAGUK proteins regulate P1AR signaling in a highly individualized manner (Hu et al. 2000; Xu et al. 2001). PSD-95 physically links PjARs to effectors, such as the N-methyl-D-aspartate (NMDA) class of glutamate receptor channels in neuronal synapses (a mechanism that is important in neurons, but not necessarily cardiomyocytes), whereas CAL redirects PjARs to the Golgi (Hu et al. 2000). In contrast, MAGI-3 (which is abundant in cardiac tissue; Wu et al. 2000) impairs PjAR coupling to the Gi-ERK pathway, but it does not grossly alter P1AR-dependent cAMP generation or agonist-dependent P1AR internalization (Hu et al. 2003; He et al. 2006). Finally, SAP97 has been implicated as a scaffold that assembles AKAP79 and PKA in complexes with P1ARs (Fig. 1a); P1AR binding to SAP79 facilitates PKA-dependent P1AR phos-phorylation at S312 in the third intracellular loop and is required for P1AR recycling following activation (Gardner et al. 2007). The observation that the WT-P1AR couples exclusively to Gs and does not undergo internalization, whereas the P1AR-PDZ mutant couples to both Gs and Gi and internalizes upon agonist exposure, has been taken as evidence that PDZ-domain-mediated interactions contribute to the control of P1AR signaling in cardiomyocytes (Xiang et al. 2002). The additional observation that SAP97 co-localizes with cadherins (cell adhesion molecules that maintain the stability of cell-cell junctions), P-catenin (which links cadherins to the actin cytoskeleton), and P1ARs at specialized postsynaptic membranes formed following cardiomyocyte sympathetic innervation suggests a mechanism for rapid responses to released neurotransmitters. However, the precise mechanism that localizes P1ARs to postsynaptic membranes remains uncertain, since a C-terminal PDZ-binding motif mutation does not disrupt P1AR localization to synapses (Shcherbakova et al. 2007).

P2ARs interact with a different set of PDZ-domain-containing proteins, namely the Na+-H+ Exchanger Regulatory Factor proteins NHERF-1 and NHERF-2 [a protein also known as Ezrin-Radixin-Moesin (ERM)-Binding Protein-50 (EBP50) that also binds to cortical actin through its ERM domain, Fig. 1, right (Hall, Premont et al. 1998; Hall et al. 1998a)]. P2AR interactions with NHERF proteins are increased by agonists and lead to enhanced Na+-H+ exchanger type 3 (NHE3) activity and P2AR recycling to surface membranes following agonist-promoted internalization (Hall et al. 1998b). In keeping with the notion that PDZ domain interactions are regulated by phosphorylations at the extreme C-terminus position -2 S/T residue, P1AR-PSD-95 and P2AR-NHERF-1 interactions are disrupted by G protein-coupled receptor kinase 5 (GRK5) phosphorylation of the PAR (Hu et al. 2002; Cao et al. 1999). P2ARs that harbor point mutations in the DSLL sequence undergo normal agonist-dependent internalization, but they do not recycle to the plasma membrane. The P2AR-DSLL sequence can also function as a transplantable sorting signal to reroute other GPCRs (that lack this extreme C-terminal sequence and are degraded following clathrin-dependent endocytosis) to a rapid recycling pathway (Gage et al. 2001).

The NHERF-binding protein ezrin plays an important role in the regulation of the actin cytoskeleton (controlling cell shape and cell adhesion/motility). Recent studies implicate ezrin as an AKAP (with greater affinity for PKA-RI, relative to RII) that modulates signaling pathways (Dransfield et al. 1997; Carlson et al. 2006). There is evidence that ezrin is a non-GPCR substrate for GRK2; GRK2-dependent ezrin-T567 phosphorylation stabilizes the active conformation that internalizes in association with P2-ARs and contributes to the control of this process (Cant and Pitcher, 2005). Ezrin also directly interacts with a polyarginine sequence (R371-R378) in the membrane-proximal portion of the a1b-AR C-tail, and this interaction between ezrin and the a1b-AR is required for a1b-AR recycling to the plasma membrane (although not internalization, Fig. 1a, Stanasila et al. 2006).

Collectively, these results identify functionally important interactions between PARs and a number of PDZ domain scaffolds that impact importantly on specific aspects of PAR signaling/desensitization. Insofar as PDZ domain scaffolding proteins tend to be expressed in a highly tissue- and cell-specific manner, these results provide a potential mechanism to explain tissue- and cell-specific differences in PAR signaling and trafficking that have been described in the literature. Moreover, these results suggest that clinical interventions designed to alter PDZ domain protein expression (or PDZ domain protein interactions with PARs or other binding partners) may have clinically important consequences that could be exploited for therapeutic advantage.

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