Research Focus

PDZ-domain arrays for identifying componentsof GPCR signaling complexes

Peter Day and Brian Kobilka

Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305, USA

Update TRENDS in Pharmacological Sciences Vol.27 No.10

Many G-protein-coupled receptors (GPCRs) modulatethe activity of multiple effectors. Yet, despite this appar-ent promiscuity, signaling in the context of differen-tiated cells is often highly specific. This specificity isattributable to the formation of cell-type-specific signal-ing complexes that are held together by scaffoldingproteins, many of which contain one or more PDZdomains. Identifying the set of potential interactionsamong GPCRs, other signaling molecules and thesescaffolding proteins is essential for understanding phy-siological signaling processes. A recent article describesan elegantly simple PDZ-domain array that can identifypotential interacting partners of GPCRs and other signal-ing molecules.

GPCR signaling complexesG-protein-coupled receptors (GPCRs) are the largestfamily of transmembrane receptors in the human genome.Individual GPCRs often couple to more than one G proteinand can signal through G-protein-independent pathways.Yet GPCR signaling and regulation in the context of dif-ferentiated cells in vivo are often specific. This specificitycan be attributed to extremely organized cell-specific sig-naling complexes that are held together by multidomainproteins linking signaling partners to each other and tospecialized domains at the plasma membrane. NumerousGPCR-interacting proteins have been identified, yet PDZ-domain-containing proteins might constitute the largestclass of proteins that is involved in organizing GCPRsignaling complexes [1].

According to the Simple Modular Architecture ResearchTool (SMART) database (http://smart.embl-heidelberg.de/),PDZ domains (a common motif that is �90 amino acids inlength) are found in 126 proteins in the human genome.These proteins have many functions, from regulating thetrafficking and targeting of proteins to assembling signal-ing complexes and networks designed for efficient andspecific signal transduction [2]. Many of these proteinscontain multiple PDZ domains, each with distinct bindingpartners. Their interactions are regulated by phosphor-ylation and competitionwith other proteins. The ligands ofPDZ domains are commonly the C-terminal three or fouramino acids of the interacting proteins, although internalligands have been documented [3]. Interactions betweenGPCRs and PDZ-domain-containing proteins have been

Corresponding author: Kobilka, B. (kobilka@stanford.edu).Available online 9 August 2006.

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identified by interaction-cloning techniques or biochem-ical approaches such as immunoprecipitation and glu-tathione-S-transferase (GST) pull-downs [4,5]. However,the relative simplicity of the recognition sequence and theabundance of PDZ domains in the human genome indicatethat all GPCRs can interact with several different PDZdomains. The ability to identify all potential interactionsbetween GPCRs and PDZ-domain-containing proteinswithin a given cell will provide insights into the composi-tion and organization of protein complexes that arerequired for physiological signaling.

PDZ-domain arraysIn two recent articles, Hall and co-workers have introduceda relatively simple approach that could have a significantimpact on efforts to understand the organization of GPCRsignaling complexes [6,7]. The investigators generated anarray of 96 class-1 PDZ domains from 50 different proteins.The PDZ domains were expressed as histidine-taggedproteins in bacteria and spotted onto nylon membranes.The array was probed with fusion proteins consisting ofGST and the C-terminal 50 amino acids of the P2Y1

receptor [7] or the last 30 amino acids of b1- and b2-adrenoceptors [6]. They identified six interactions withb1-adrenoceptors. Four of these had been reported pre-viously [those with postsynaptic density protein of 95 kDa,membrane-associated guanylate kinase inverted (MAGI)-2, conductance regulator-associated ligand and GAIP-interacting protein, C-terminal] and two novel interactionswere identified (those with SAP97 and MAGI-3). Theauthors demonstrated the specificity of the array by show-ing that theGST fusions containing theC-termini of the b2-adrenoceptor, b1-adrenoceptor or P2Y1 receptor bind todifferent sets of PDZ domain [6,7]. The PDZ-domain inter-actions identified on the array with the C-terminus of theb2-adrenoceptor had previously been identified; however, anovel interaction was detected between the second PDZdomain of Na+–H+ exchanger regulatory factor 2 and theP2Y1 receptor tail [6,7].

The interactions between the b1-adrenoceptor andMAGI-3 were detected when co-expressed in HEK-293cells. Of interest, MAGI-3 localized in the nucleus in theabsence of b1-adrenoceptors, but targeted to the plasmamembrane when both were expressed in HEK-293 cells.MAGI-3 had no effect on the b1-adrenoceptor activation ofadenylyl cyclase or the agonist-induced internalization ofthe b1-adrenoceptor, but it inhibited the b1-adrenoceptoractivation of extracellular-signal-regulated kinase (ERK)in Cos-7 cells.

d. doi:10.1016/j.tips.2006.08.003

510 Update TRENDS in Pharmacological Sciences Vol.27 No.10

Potential role of MAGI-3 in b1-adrenoceptor signaling inthe heartBoth SAP97 and MAGI-3 are expressed in cardiac myo-cytes, which raises questions about which interaction isfunctionally the most relevant. PDZ-domain interactionsare important for the physiological function of both b1- andb2-adrenoceptors in neonatal cardiac myocytes [8,9]. Theb1-adrenoceptor does not undergo internalization and cou-ples only to Gas in these myocytes [10]. Disruption of thePDZ ligand at the C-terminus of the b1-adrenoceptor (b1-adrenoceptor–PDZ) leads to a significant increase in ago-nist-induced internalization and promotes coupling to Gai

[8]. Little is known about the function of MAGI-3 in theheart; however, it is of interest that He et al. [6] found thatMAGI-3 prevents coupling of the b1-adrenoceptor to ERKactivation in Cos-7 cells, which is a Gai-mediated process.This could explain the signaling behavior of the b1-adre-noceptor–PDZ mutant in cardiac myocytes.

MAGI-3 contains five type-1 PDZ domains, and the b1-adrenoceptor binds to PDZ domain 1. Identifying proteinsthat bind to the other PDZ domains could provide clues tothe role of MAGI-3 in b1-adrenoceptor signaling. MAGI-3was initially cloned from a yeast two-hybrid screen toidentify regulators of the phosphatase with tensinhomology mutated in multiple advanced cancers(PTEN/MMAC) [11]. This enzyme catalyzes theremoval of the 3-phosphate from phosphatidylinositol(3,4,5)-trisphosphate [PtdIns(3,4,5)P3], which is anactivator of the protein product of the cellular Akt geneor protein kinase B (Akt/PKB)–a serine/threonine kinasethat, among other actions, suppresses apoptosis. By depho-sphorylating PtdIns(3,4,5)P3, PTEN/MMAC suppressesthe activation of Akt/PKB. This is of interest becausethe activation of PtdIns 3-kinase and Akt is common tomany cardioprotective agents, including insulin and insu-lin-like growth factor-1 [12]. This pathway has also beenimplicated in the cardioprotective effects of b2-adrenocep-tor activation [13]. Wu et al. [11] demonstrated that MAGI-3 enhances the suppression of Akt by PTEN/MMAC, whichbinds to PDZ domain 2 of MAGI-3; thus, the b1-adreno-ceptor–MAGI-3 complex might localize PTEN/MMAC toits substrate in the plasmamembrane of cardiac myocytes.This mechanism could contribute to the pro-apoptoticeffects of prolonged b1-adrenoceptor stimulation.

MAGI-3 also binds to frizzled receptors 4 and 7; both ofthese seven-transmembrane receptors are expressed in theheart and might have a role in cardiac remodeling inresponse to injury [14]. As is the case with b1-adrenocep-tors, both frizzled receptor subtypes bind to PDZ domain 1of MAGI-3. Thus, depending on the relative abundance ofthe different proteins, there might be competition amongthese GPCRs for a limited supply of MAGI-3, or MAGI-3might have roles in the signaling of all three receptors.

Concluding remarksThis newly identified interaction between b1-adrenocep-tors and MAGI-3, together with the knowledge of otherproteins that interact with MAGI-3, enables the genera-tion of testable hypotheses regarding new signaling andregulatory pathways. This example illustrates the poten-tial of this proteomics approach for identifying

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physiologically important interactions between signalingmolecules. However, this new proteomics tool is not with-out limitations. The binding of C-terminal peptides toisolated PDZ domains does not account for secondaryinteractions that could contribute to binding affinity andspecificity. Moreover, PDZ-domain interactions are notlimited to C-terminal sequences. Finally, it is possible thatnot all interactions identified will be physiologically rele-vant. Expression profiling will be required to identifyinteracting partners expressed together in vivo.

Nevertheless, this simple array has the potential toprovide an unbiased survey of all possible interactionswith both membrane-bound and cytosolic signaling pro-teins. The arrayhas the additional advantage of being ableto identify interactions with a specific PDZ domain(or domains) in a protein that contains several suchdomains. In this way, it is possible to identify potentiallinks between multiple signaling molecules. The arrayformat developed by Hall and colleagues can be readilyapplied to virtually all GPCRs, without the need forsophisticated array-generating or -reading devices.

Recently, Stiffler et al. [15] developed amore technicallycomplex array consisting of a 9 � 9 matrix of PDZ domainsin eachwell of a 96-well plate. The array can be probedwithlibraries of fluorescent peptides to identify specific protein–protein interactions. This approach requires more-specia-lized equipment to generate and read the arrays; however,it can screen larger arrays of peptides to determine thespectrum of sequences that can be recognized by each PDZdomain. The impact of these new proteomics tools onGPCRbiology could be significant if the arrays were made avail-able to all interested investigators and if the data obtainedfrom these studies could be readily accessed from a centraldatabase.

References1 Bockaert, J. et al. (2004) GPCR interacting proteins (GIP). Pharmacol.

Ther. 103, 203–2212 Nourry, C. et al. (2003) PDZ domain proteins: plug and play! Sci. STKE

2003, RE73 Hillier, B.J. et al. (1999) Unexpected modes of PDZ domain scaffolding

revealed by structure of nNOS–syntrophin complex. Science 284, 812–815

4 Hu, L.A. et al. (2000) b1-adrenergic receptor association with PSD-95.Inhibition of receptor internalization and facilitation of b1-adrenergicreceptor interaction with N-methyl-D-aspartate receptors. J. Biol.Chem. 275, 38659–38666

5 Xu, J. et al. (2001) b1-adrenergic receptor association with the synapticscaffolding protein membrane-associated guanylate kinase inverted-2(MAGI-2). Differential regulation of receptor internalization by MAGI-2 and PSD-95. J. Biol. Chem. 276, 41310–41317

6 He, J. et al. (2006) Proteomic analysis of b1-adrenergic receptorinteractions with PDZ scaffold proteins. J. Biol. Chem. 281, 2820–2827

7 Fam, S.R. et al. (2005) P2Y1 receptor signaling is controlled byinteraction with the PDZ scaffold NHERF-2. Proc. Natl. Acad. Sci.U. S. A. 102, 8042–8047

8 Xiang, Y. et al. (2002) The PDZ binding motif of the b1 adrenergicreceptor modulates receptor trafficking and signaling in cardiacmyocytes. J. Biol. Chem. 277, 33783–33790

9 Xiang, Y. and Kobilka, B. (2003) The PDZ-binding motif of theb2-adrenoceptor is essential for physiologic signaling andtrafficking in cardiac myocytes. Proc. Natl. Acad. Sci. U. S. A. 100,10776–10781

10 Devic, E. et al. (2001) b-adrenergic receptor subtype-specific signalingin cardiac myocytes from b1 and b2 adrenoceptor knockout mice. Mol.Pharmacol. 60, 577–583

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11 Wu, Y. et al. (2000) Interaction of the tumor suppressor PTEN/MMACwith a PDZ domain ofMAGI3, a novel membrane-associated guanylatekinase. J. Biol. Chem. 275, 21477–21485

12 Matsui, T. and Rosenzweig, A. (2005) Convergent signal transductionpathways controlling cardiomyocyte survival and function: the role ofPI 3-kinase and Akt. J. Mol. Cell. Cardiol. 38, 63–71

13 Chesley, A. et al. (2000) The b2-adrenergic receptor delivers anantiapoptotic signal to cardiac myocytes through Gi-dependent

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14 Yao, R. et al. (2004) MAGI-3 is involved in the regulation of the JNKsignaling pathway as a scaffold protein for frizzled and Ltap. Oncogene23, 6023–6030

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