PDZ Domains: Structural Modules for Protein Complex Assembly

Abstract

PDZ domains 1PDZPSD-95/Dlg/ZO-1 homologyPSDpostsynaptic densitySAPsynapse-associated proteinNMDAN-methyl-d-aspartateNHERFNa+/H+ exchange regulatory factorSH2Src homology 2SH3Src homology 3nNOSneuronal nitric-oxide synthaseCFTRcystic fibrosis transmembrane regulatorPICK1protein interacting with C-kinase 1AMPAα-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acidGRIPglutamate receptor-interacting proteinInaDinactivation-no afterpotential DMUPP1multi-PDZ containing protein 1MAGUKmembrane-associated guanylate kinase 1PDZPSD-95/Dlg/ZO-1 homologyPSDpostsynaptic densitySAPsynapse-associated proteinNMDAN-methyl-d-aspartateNHERFNa+/H+ exchange regulatory factorSH2Src homology 2SH3Src homology 3nNOSneuronal nitric-oxide synthaseCFTRcystic fibrosis transmembrane regulatorPICK1protein interacting with C-kinase 1AMPAα-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acidGRIPglutamate receptor-interacting proteinInaDinactivation-no afterpotential DMUPP1multi-PDZ containing protein 1MAGUKmembrane-associated guanylate kinase are modular protein interaction domains that play a role in protein targeting and protein complex assembly. Once termed Discs-large homology regions (DHRs) or GLGF repeats (after a conserved Gly-Leu-Gly-Phe sequence found within the domain), these domains of ∼90 amino acids are now primarily known by an acronym of the first three PDZ-containing proteins identified: the postsynaptic protein PSD-95/SAP90, theDrosophila septate junction protein Discs-large, and the tight junction protein ZO-1. Since their initial identification, PDZ and PDZ-like domains have been recognized in numerous proteins from organisms as diverse as bacteria, plants, yeast, metazoans, and Drosophila (1Ponting C.P. Protein Sci. 1997; 6: 464-468Crossref PubMed Scopus (189) Google Scholar). In fact, they are among the commonest protein domains represented in sequenced genomes. Analysis of the human, Drosophila, and Caenorhabditis elegansgenomes estimates the presence of 440 PDZ domains in 259 different proteins, 133 PDZ domains in 86 proteins, and 138 PDZ domains in 96 proteins, respectively (2Schultz J. Copley R.R. Doerks T. Ponting C.P. Bork P. Nucleic Acids Res. 2000; 28: 231-234Crossref PubMed Scopus (1018) Google Scholar). PSD-95/Dlg/ZO-1 homology postsynaptic density synapse-associated protein N-methyl-d-aspartate Na+/H+ exchange regulatory factor Src homology 2 Src homology 3 neuronal nitric-oxide synthase cystic fibrosis transmembrane regulator protein interacting with C-kinase 1 α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid glutamate receptor-interacting protein inactivation-no afterpotential D multi-PDZ containing protein 1 membrane-associated guanylate kinase PSD-95/Dlg/ZO-1 homology postsynaptic density synapse-associated protein N-methyl-d-aspartate Na+/H+ exchange regulatory factor Src homology 2 Src homology 3 neuronal nitric-oxide synthase cystic fibrosis transmembrane regulator protein interacting with C-kinase 1 α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid glutamate receptor-interacting protein inactivation-no afterpotential D multi-PDZ containing protein 1 membrane-associated guanylate kinase The structural features of PDZ domains allow them to mediate specific protein-protein interactions that underlie the assembly of large protein complexes involved in signaling or subcellular transport. Not surprisingly, disrupting these interactions can play a role in human diseases. Mutations in a gene encoding harmonin, a PDZ-containing protein, cause Usher syndrome type 1C, an autosomal recessive disorder characterized by congenital sensorineural deafness, vestibular dysfunction, and blindness (3Verpy E. Leibovici M. Zwaenepoel I. Liu X.Z. Gal A. Salem N. Mansour A. Blanchard S. Kobayashi I. Keats B.J. Slim R. Petit C. Nat. Genet. 2000; 26: 51-55Crossref PubMed Scopus (366) Google Scholar, 4Bitner-Glindzicz M. Lindley K.J. Rutland P. Blaydon D. Smith V.V. Milla P.J. Hussain K. Furth-Lavi J. Cosgrove K.E. Shepherd R.M. Barnes P.D. O'Brien R.E. Farndon P.A. Sowden J. Liu X.Z. Scanlan M.J. Malcolm S. Dunne M.J. Aynsley-Green A. Glaser B. Nat. Genet. 2000; 26: 56-60Crossref PubMed Scopus (251) Google Scholar, 5Montell C.J. Nat. Genet. 2000; 26: 6-7Crossref PubMed Scopus (13) Google Scholar). This was the first mutation in a PDZ-encoding gene linked to a human disease. Subsequently, mutations in the periaxin gene, which also encodes a PDZ-containing protein, have been identified as a cause of Dejerine-Sottas neuropathy, a severe demyelinating form of peripheral neuropathy (6Boerkoel C.F. Takashima H. Stankiewicz P. Garcia C.A. Leber S.M. Rhee-Morris L. Lupski J.R. Am. J. Hum. Genet. 2001; 68: 325-333Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar, 7Sherman D.L. Fabrizi C. Gillespie C.S. Brophy P.J. Neuron. 2001; 30: 677-687Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). The notion that PDZ domains serve as protein interaction modules emerged from the finding that the first and second PDZ (PDZ1 and -2) domains of PSD-95 can bind the extreme C-terminal peptide sequence of Shaker-type K+ channels (8Kim E. Niethammer M. Rothschild A. Jan Y.N. Sheng M. Nature. 1995; 378: 85-88Crossref PubMed Scopus (892) Google Scholar) and NMDA receptor NR2 subunits (9Kornau H.-C. Schenker L.T. Kennedy M.B. Seeburg P.H. Science. 1995; 269: 1737-1740Crossref PubMed Scopus (1611) Google Scholar, 10Niethammer M. Kim E. Sheng M. J. Neurosci. 1996; 16: 2157-2163Crossref PubMed Google Scholar). PDZ3 of the protein tyrosine phosphatase FAP-1/PTP1E similarly was identified as a binding site for the C terminus of the cell surface receptor Fas (11Sato T. Irie S. Kitada S. Reed J.C. Science. 1995; 268: 411-415Crossref PubMed Scopus (691) Google Scholar). These studies further demonstrated that PDZ domains maintained their activity and selectivity when expressed in heterologous proteins, establishing these motifs as modular domains that bind the C termini of target proteins in a sequence-specific manner. The structural basis for PDZ specificity became apparent with the solution of the x-ray crystallographic structure of PDZ domains complexed with their cognate peptide ligands. First solved for PDZ3 of PSD-95 (12Doyle D.A. Lee A. Lewis J. Kim E. Sheng M. MacKinnon R. Cell. 1996; 85: 1067-1076Abstract Full Text Full Text PDF PubMed Scopus (953) Google Scholar, 13Morais Cabral J.H. Petosa C. Sutcliffe M.J. Raza S. Byron O. Poy F. Marfatia S.M. Chishti A.H. Liddington R.C. Nature. 1996; 382: 649-652Crossref PubMed Scopus (286) Google Scholar), numerous additional PDZ crystal structures have been determined in recent years, including PDZ2 of PSD-95 (14Tochio H. Hung F., Li, M. Bredt D.S. Zhang M. J. Mol. Biol. 2000; 295: 225-237Crossref PubMed Scopus (91) Google Scholar), the single PDZ domain of CASK (15Daniels D.L. Cohen A.R. Anderson J.M. Brunger A.T. Nat. Struct. Biol. 1998; 5: 317-325Crossref PubMed Scopus (160) Google Scholar), syntrophin (16Schultz J. Hoffmuller U. Krause G. Ashurst J. Macias M.J. Schmieder P. Schneider-Mergener J. Oschkinat H. Nat. Struct. Biol. 1998; 5: 19-24Crossref PubMed Scopus (202) Google Scholar) and neuronal nitric-oxide synthase (nNOS) (17Hillier B.J. Christopherson K.S. Prehoda K.E. Bredt D.S. Lim W.A. Science. 1999; 284: 812-815Crossref PubMed Scopus (461) Google Scholar), PDZ2 from human phosphatase hPTP1E (18Kozlov G. Gehring K. Ekiel I. Biochemistry. 2000; 39: 2572-2580Crossref PubMed Scopus (60) Google Scholar), PDZ1 of the Na+/H+ exchanger regulatory factor (NHERF) (19Karthikeyan S. Leung T. Ladias J.A. J. Biol. Chem. 2001; 276: 19683-19686Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar), and PDZ1 of InaD (20Kimple M.E. Siderovski D.P. Sondek J. EMBO J. 2001; 20: 4414-4422Crossref PubMed Scopus (43) Google Scholar). The common structure of PDZ domains comprises six β strands (βA–βF) and two α helices (αA and αB), which fold in an overall six-stranded β sandwich (Fig.1 A). C-terminal peptides bind as an antiparallel β-strand in a groove between the βB strand and the αB helix, in essence extending one of the β-sheets. The conserved Gly-Leu-Gly-Phe (GLGF) sequence of the PDZ domain is found within the βA–βB connecting loop and is important for hydrogen bond coordination of the C-terminal carboxylate (COO−) group. The N and C termini of the PDZ domain are located near each other on the opposite side of the PDZ domain from the peptide-binding site, a feature shared with other protein interaction modules such as SH2 domains. PDZ-like domains identified in plants and bacteria have a similar overall secondary and tertiary structure but show a different topology (Fig. 1 C). Specifically, the βA strand, derived from the N-terminal sequences in conventional PDZ domains, is formed by the C terminus in the PDZ-like domain of the photosystem II D1 C-terminal protease (21Liao D. Qian J. Chisholm D.A. Jordan D.B. Diner B.A. Nat. Struct. Biol. 2000; 7: 749-753Crossref PubMed Scopus (111) Google Scholar). This circularly permuted fold, also found in the Tsp protease from Escherichia coli, retains the ability to bind C-terminal sequences (22Beebe K.D. Shin J. Peng J. Chaudhury C. Khera J. Pei D. Biochemistry. 2000; 39: 3149-3155Crossref PubMed Scopus (72) Google Scholar). The crystallographic data indicate that the C-terminal four residues of PDZ ligands interact directly with the peptide-binding groove. The main chain atoms of the βB strand form hydrogen bonds with the extended peptide ligand and stabilize the interaction although they do not account for sequence specificity. The specific interaction of C-terminal sequences suggests that recognition of the carboxylate group is critical for PDZ binding. Indeed, a highly conserved positively charged residue (e.g. arginine 318 of PDZ3 of PSD-95) and the main chain amides of the Gly-Leu-Gly-Phe motif form hydrogen bonds with the terminal carboxylate group (12Doyle D.A. Lee A. Lewis J. Kim E. Sheng M. MacKinnon R. Cell. 1996; 85: 1067-1076Abstract Full Text Full Text PDF PubMed Scopus (953) Google Scholar). The side chain of the C-terminal residue (position 0) projects into a hydrophobic pocket accounting for PDZ domains binding preferentially to sequences ending with a hydrophobic residue (such as valine, isoleucine, or leucine (23Songyang Z. Fanning A.S., Fu, C., Xu, J. Marfatia S.M. Chishti A.H. Crompton A. Chan A.C. Anderson J.M. Cantley L.C. Science. 1997; 275: 73-77Crossref PubMed Scopus (1209) Google Scholar)). In the crystal structure of PSD-95 PDZ3 with its peptide ligand, the side chain of the residue at position −1 points away from the interaction surface (12Doyle D.A. Lee A. Lewis J. Kim E. Sheng M. MacKinnon R. Cell. 1996; 85: 1067-1076Abstract Full Text Full Text PDF PubMed Scopus (953) Google Scholar). This correlates with the relative lack of PDZ specificity for recognition at the −1 position. Nevertheless, substitutions at this site can affect binding preference for individual PDZ domains, albeit to a lesser degree than the 0 and −2 positions (23Songyang Z. Fanning A.S., Fu, C., Xu, J. Marfatia S.M. Chishti A.H. Crompton A. Chan A.C. Anderson J.M. Cantley L.C. Science. 1997; 275: 73-77Crossref PubMed Scopus (1209) Google Scholar, 24Stricker N.L. Christopherson K.S. Yi B.A. Schatz P.J. Raab R.W. Dawes G. Bassett D.E. Bredt D.S. Li M. Nat. Biotechnol. 1997; 15: 336-342Crossref PubMed Scopus (218) Google Scholar, 25Niethammer M. Valtschanoff J.G. Kapoor T.M. Allison D.W. Weinberg T.M. Craig A.M. Sheng M. Neuron. 1998; 20: 693-707Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar). In contrast, the guanido group of arginine at position −1 of the cystic fibrosis transmembrane regulator (CFTR) C terminus (C-terminal sequence -DTRL) forms two salt bridges and two hydrogen bonds with residues in PDZ1 of NHERF, indicating that in some cases the −1 residue contributes directly to the specificity and affinity of the interaction (19Karthikeyan S. Leung T. Ladias J.A. J. Biol. Chem. 2001; 276: 19683-19686Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). The crystal structure of the InaD PDZ1 with a C-terminal peptide from NorpA (C-terminal sequence -EFCA) further demonstrates a critical role for a cysteine residue at position −1, as intermolecular disulfide bond formation of this residue with a cysteine residue in the PDZ domain is required for high affinity interaction (20Kimple M.E. Siderovski D.P. Sondek J. EMBO J. 2001; 20: 4414-4422Crossref PubMed Scopus (43) Google Scholar). The binding specificity of PDZ domains is critically determined by the interaction of the first residue of helix αB (position αB1) and the side chain of the −2 residue of the C-terminal ligand; this forms the basis for PDZ classification (Table I) (23Songyang Z. Fanning A.S., Fu, C., Xu, J. Marfatia S.M. Chishti A.H. Crompton A. Chan A.C. Anderson J.M. Cantley L.C. Science. 1997; 275: 73-77Crossref PubMed Scopus (1209) Google Scholar). In Class I PDZ interactions, such as those of PSD-95, a serine or threonine residue occupies the −2 position. The side chain hydroxyl group forms a hydrogen bond with the N-3 nitrogen of a histidine residue at position αB1 (12Doyle D.A. Lee A. Lewis J. Kim E. Sheng M. MacKinnon R. Cell. 1996; 85: 1067-1076Abstract Full Text Full Text PDF PubMed Scopus (953) Google Scholar), which is highly conserved among Class I PDZ domains. In contrast, class II PDZ interactions are characterized by hydrophobic residues at both the −2 position of the peptide ligand and the αB1 position of the PDZ domain (23Songyang Z. Fanning A.S., Fu, C., Xu, J. Marfatia S.M. Chishti A.H. Crompton A. Chan A.C. Anderson J.M. Cantley L.C. Science. 1997; 275: 73-77Crossref PubMed Scopus (1209) Google Scholar). A third class of PDZ domains, such as nNOS, prefers negatively charged amino acids at the −2 position (24Stricker N.L. Christopherson K.S. Yi B.A. Schatz P.J. Raab R.W. Dawes G. Bassett D.E. Bredt D.S. Li M. Nat. Biotechnol. 1997; 15: 336-342Crossref PubMed Scopus (218) Google Scholar). This specificity is determined by the coordination of the hydroxyl group of a tyrosine residue at position αB1 with the side chain carboxylate of the −2 residue (24Stricker N.L. Christopherson K.S. Yi B.A. Schatz P.J. Raab R.W. Dawes G. Bassett D.E. Bredt D.S. Li M. Nat. Biotechnol. 1997; 15: 336-342Crossref PubMed Scopus (218) Google Scholar, 26Tochio H. Zhang Q. Mandal P., Li, M. Zhang M. Nat. Struct. Biol. 1999; 6: 417-421Crossref PubMed Scopus (136) Google Scholar). Other classes of PDZ domain specificities are likely to be distinguished with further research.Table IExamples of PDZ domains classified by specificity for C-terminal peptide ligandsClassC-terminal sequence1-aLast four amino acids of the ligand proteins are shown although specificity can involve more proximal residues as well (see text). Φ, hydrophobic amino acid;X, unspecified amino acid.Ligand proteinPDZ proteinRef.Class I -X-S/T-X-Φ-ETDVShaker K+ channelPSD-95 (PDZ2)8Kim E. Niethammer M. Rothschild A. Jan Y.N. Sheng M. Nature. 1995; 378: 85-88Crossref PubMed Scopus (892) Google Scholar-ESDVNMDA receptor subunits NR2A/BPSD-95 (PDZ2)9Kornau H.-C. Schenker L.T. Kennedy M.B. Seeburg P.H. Science. 1995; 269: 1737-1740Crossref PubMed Scopus (1611) Google Scholar-TTRVNeuroliginPSD-95 (PDZ3)68Irie M. Hata Y. Takeuchi M. Ichtchenko K. Toyoda A. Hirao K. Takai Y. Rosahl T.W. Sudhof T.C. Science. 1997; 277: 1511-1515Crossref PubMed Scopus (594) Google Scholar-ESLVVoltage-gated Na+ channelSyntrophin69Gee S.H. Madhavan R. Levinson S.R. Caldwell J.H. Sealock R. Froehner S.C. J. Neurosci. 1998; 18: 128-137Crossref PubMed Google Scholar-QSAVProtein kinase C-αPICK127Staudinger J., Lu, J. Olson E.N. J. Biol. Chem. 1997; 272: 32019-32024Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar-DSSLβ2-Adrenergic receptorNHERF (PDZ1)35Hall R.A. Premont R.T. Chow C.W. Blitzer J.T. Pitcher J.A. Claing A. Stoffel R.H. Barak L.S. Shenolikar S. Weinman E.J. Grinstein S. Lefkowitz R.J. Nature. 1998; 392: 626-630Crossref PubMed Scopus (516) Google Scholar-DTRLCFTRNHERF (PDZ1)70Wang S. Raab R.W. Schatz P.J. Guggino W.B. Li M. FEBS Lett. 1998; 427: 103-108Crossref PubMed Scopus (247) Google Scholar-QTRLGKAPShank/ProSAP71Naisbitt S. Kim E., Tu, J.C. Xiao B. Sala C. Valtschanoff J. Weinberg R.J. Worley P.F. Sheng M. Neuron. 1999; 23: 569-582Abstract Full Text Full Text PDF PubMed Scopus (769) Google Scholar-SSTLMetabotropic glutamate receptor subunit mGluR5Shank/ProSAP72Tu J.C. Xiao B. Naisbitt S. Yuan J.P. Petralia R.S. Brakeman P. Doan A. Aakalu V.K. Lanahan A.A. Sheng M. Worley P.F. Neuron. 1999; 23: 583-592Abstract Full Text Full Text PDF PubMed Scopus (876) Google ScholarClass II -X-Φ-X-Φ-SVKIAMPA receptor subunit GluR2PICK128Xia J. Zhang X. Staudinger J. Huganir R.L. Neuron. 1999; 22: 179-187Abstract Full Text Full Text PDF PubMed Scopus (487) Google ScholarGRIP (PDZ5)39Dong H. Zhang P. Song I. Petralia R.S. Liao D. Huganir R.L. J. Neurosci. 1999; 19: 6930-6941Crossref PubMed Google Scholar-EYFIGlycophorin CErythrocyte p5573Marfatia S.M. Morais-Cabral J.H. Kim A.C. Byron O. Chishti A.H. J. Biol. Chem. 1997; 272: 24191-24197Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar-EYYVNeurexinCASK74Hata Y. Butz S. Sudhof T.C. J. Neurosci. 1996; 16: 2488-2494Crossref PubMed Google Scholar-EFYASyndecan-2CASK75Hsueh Y.-P. Yang F.-C. Kharazia V. Naisbitt S. Cohen A.R. Weinberg R.J. Sheng M. J. Cell Biol. 1998; 142: 139-151Crossref PubMed Scopus (283) Google Scholar-YYKVEphrin B1PICK176Torres R. Firestein B.L. Dong H. Staudinger J. Olson E.N. Huganir R.L. Bredt D.S. Gale N.W. Yancopoulos G.D. Neuron. 1998; 21: 1453-1463Abstract Full Text Full Text PDF PubMed Scopus (395) Google ScholarGRIP (PDZ6)Syntenin (PDZ2)Class III -X-D/E-X-Φ-VDSVMelatonin receptornNOS24Stricker N.L. Christopherson K.S. Yi B.A. Schatz P.J. Raab R.W. Dawes G. Bassett D.E. Bredt D.S. Li M. Nat. Biotechnol. 1997; 15: 336-342Crossref PubMed Scopus (218) Google Scholar-GEPLKIF17mLIN10/Mint1/X1164Setou M. Nakagawa T. Seog D.H. Hirokawa N. Science. 2000; 288: 1796-1802Crossref PubMed Scopus (597) Google Scholar1-a Last four amino acids of the ligand proteins are shown although specificity can involve more proximal residues as well (see text). Φ, hydrophobic amino acid;X, unspecified amino acid. Open table in a new tab PDZ domains vary in their range and stringency of specificity. For example, the PDZ domain of PICK1, which has a lysine residue at the αB1 position, can bind the C termini of both protein kinase C (ending in -QSAV (27Staudinger J., Lu, J. Olson E.N. J. Biol. Chem. 1997; 272: 32019-32024Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar)) and the AMPA receptor subunit GluR2 (ending in -SVKI (28Xia J. Zhang X. Staudinger J. Huganir R.L. Neuron. 1999; 22: 179-187Abstract Full Text Full Text PDF PubMed Scopus (487) Google Scholar)). Thus, a single PDZ domain can show both Class I and Class II specificity; the three-dimensional structural basis of this promiscuity remains to be determined. Whereas the residue at the −2 position is a key determinant of PDZ-ligand interactions, more N-terminal residues in PDZ-binding peptides also contribute to specificity. Crystallographic data indicate that the −3 side chain also directly contacts the peptide-binding groove (12Doyle D.A. Lee A. Lewis J. Kim E. Sheng M. MacKinnon R. Cell. 1996; 85: 1067-1076Abstract Full Text Full Text PDF PubMed Scopus (953) Google Scholar, 19Karthikeyan S. Leung T. Ladias J.A. J. Biol. Chem. 2001; 276: 19683-19686Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 26Tochio H. Zhang Q. Mandal P., Li, M. Zhang M. Nat. Struct. Biol. 1999; 6: 417-421Crossref PubMed Scopus (136) Google Scholar), and the residue at position −3 is important in determining the binding of ligands selected from a peptide library (23Songyang Z. Fanning A.S., Fu, C., Xu, J. Marfatia S.M. Chishti A.H. Crompton A. Chan A.C. Anderson J.M. Cantley L.C. Science. 1997; 275: 73-77Crossref PubMed Scopus (1209) Google Scholar). Moreover, several studies have demonstrated that residues beyond the last four amino acids are also important, up to position −8 (18Kozlov G. Gehring K. Ekiel I. Biochemistry. 2000; 39: 2572-2580Crossref PubMed Scopus (60) Google Scholar,23Songyang Z. Fanning A.S., Fu, C., Xu, J. Marfatia S.M. Chishti A.H. Crompton A. Chan A.C. Anderson J.M. Cantley L.C. Science. 1997; 275: 73-77Crossref PubMed Scopus (1209) Google Scholar, 25Niethammer M. Valtschanoff J.G. Kapoor T.M. Allison D.W. Weinberg T.M. Craig A.M. Sheng M. Neuron. 1998; 20: 693-707Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar). Although many examples of PDZ-peptide ligand interactions have now been identified, uncertainty remains regarding the binding affinity of the PDZ domain for its ligand. Using solid phase methods such as surface plasmon resonance (Biacore) or modified enzyme-linked immunosorbent assays, affinities of PDZ binding to their cognate peptides have been measured in the 10–100 nm range. On the other hand, solution methods such as fluorescence polarization suggest that the binding affinity may be weaker in the low micromolar range (25Niethammer M. Valtschanoff J.G. Kapoor T.M. Allison D.W. Weinberg T.M. Craig A.M. Sheng M. Neuron. 1998; 20: 693-707Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar, 29Harris B.Z. Hillier B.J. Lim W.A. Biochemistry. 2001; 40: 5921-5930Crossref PubMed Scopus (104) Google Scholar). It is likely that a wide range of affinities applies to the diversity of PDZ domain interactions in vivo. Moreover, especially because many PDZ ligands are membrane-associated and clustered, it is difficult to know whether solid phase or solution methods better approach the in vivo situation. The regulation of protein-protein interactions between modular elements such as SH3 domains and their binding partners is often critical for cell signaling (30Pawson T. Scott J.D. Science. 1997; 278: 2075-2080Crossref PubMed Scopus (1883) Google Scholar). What regulates the binding of PDZ domains to their respective C-terminal ligands? One mode of control is the phosphorylation of residues within the C-terminal sequences that bind the PDZ domain. For example, serine phosphorylation at position −2 in the inward rectifier K+ channel Kir2.3 by protein kinase A disrupts binding to the PDZ domains of PSD-95 (31Cohen N.A. Brenman J.E. Snyder S. Bredt D.S. Neuron. 1996; 17: 759-767Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar). The association of β2-adrenergic receptor with NHERF is similarly abolished by phosphorylation at position −2 by G-protein-coupled receptor kinase GRK5 (32Cao T.T. Deacon H.W. Reczek D. Bretscher A. van Zastrow M. Nature. 1999; 401: 286-290Crossref PubMed Scopus (561) Google Scholar). Intriguingly, binding of a single C-terminal peptide ligand to multiple PDZ domains can be differentially regulated. For instance, phosphorylation at serine 880 of the AMPA receptor subunit GluR2 (C-terminal sequence -SVKI) by protein kinase C inhibits binding to the PDZ domain of GRIP1 but not to PICK1 (33Matsuda S. Mikawa S. Hirai H. J. Neurochem. 1999; 73: 1765-1768Crossref PubMed Scopus (226) Google Scholar, 34Chung H.J. Xia J. Scannevin R.H. Zhang X. Huganir R.L. J. Neurosci. 2000; 20: 7258-7267Crossref PubMed Google Scholar). PDZ interactions may also be regulated by extracellular signals, as the binding of the β2-adrenergic receptor C terminus to NHERF is stimulated by β-adrenergic agonists (35Hall R.A. Premont R.T. Chow C.W. Blitzer J.T. Pitcher J.A. Claing A. Stoffel R.H. Barak L.S. Shenolikar S. Weinman E.J. Grinstein S. Lefkowitz R.J. Nature. 1998; 392: 626-630Crossref PubMed Scopus (516) Google Scholar). Thus, it is likely that both intracellular and extracellular signals regulate the temporal and spatial organization of PDZ-based interactions. Although binding to C-terminal peptides appears to be the typical mode of interaction, PDZ domains can also interact with internal peptide sequences. The best example of this is the interaction of nNOS with the PDZ domain of PSD-95 or syntrophin (36Brenman J.E. Chao D.S. Gee S.H. McGee A.W. Craven S.E. Santillano D.R., Wu, Z. Huang F. Xia H. Peters M.F. Froehner S.C. Bredt D.S. Cell. 1996; 84: 757-767Abstract Full Text Full Text PDF PubMed Scopus (1426) Google Scholar, 37Brenman J.E. Christopherson K.S. Craven S.E. McGee A.W. Bredt D.S. J. Neurosci. 1996; 16: 7407-7415Crossref PubMed Google Scholar). In the crystal structure of the nNOS-syntrophin PDZ complex, amino acid residues adjacent to the canonical PDZ domain of nNOS form a two-stranded β-hairpin “finger,” which docks in the peptide-binding groove of the syntrophin PDZ domain (Fig. 1 B) (14Tochio H. Hung F., Li, M. Bredt D.S. Zhang M. J. Mol. Biol. 2000; 295: 225-237Crossref PubMed Scopus (91) Google Scholar, 26Tochio H. Zhang Q. Mandal P., Li, M. Zhang M. Nat. Struct. Biol. 1999; 6: 417-421Crossref PubMed Scopus (136) Google Scholar). The sharp β turn of the β-finger binds to the same site as the terminal carboxylate group of peptide ligands (36Brenman J.E. Chao D.S. Gee S.H. McGee A.W. Craven S.E. Santillano D.R., Wu, Z. Huang F. Xia H. Peters M.F. Froehner S.C. Bredt D.S. Cell. 1996; 84: 757-767Abstract Full Text Full Text PDF PubMed Scopus (1426) Google Scholar). Introduction of point mutations that destabilize the nNOS β-finger conformation results in decreased binding to the syntrophin PDZ domain, supporting the model that the β-finger is required for proper recognition (29Harris B.Z. Hillier B.J. Lim W.A. Biochemistry. 2001; 40: 5921-5930Crossref PubMed Scopus (104) Google Scholar). Screening of combinatorial phage libraries has also identified cyclic peptides as potential ligands for PDZ domains; PDZ binding of these peptides depends on intramolecular disulfide bond formation (38Gee S.H. Sekely S.A. Lombardo C. Kurakin A. Froehner S.C. Kay B.K. J. Biol. Chem. 1998; 273: 21980-21987Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). These examples suggest that PDZ domains may interact with internal sequences that are conformationally constrained, structurally mimicking a free C terminus. In addition, PDZ domains can associate with other PDZ domains to form homo- and hetero-oligomers. Examples include GRIP (39Dong H. Zhang P. Song I. Petralia R.S. Liao D. Huganir R.L. J. Neurosci. 1999; 19: 6930-6941Crossref PubMed Google Scholar, 40Srivastava S. Osten P. Vilim F.S. Khatri L. Inman G. States B. Daly C. DeSouza S. Abagyan R. Valtschanoff J.G. Weinberg R.J. Ziff E.B. Neuron. 1998; 21: 581-591Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar), ZO-1 (41Fanning A.S. Jameson B.J. Jesaitis L.A. Anderson J.M. J. Biol. Chem. 1998; 273: 29745-29753Abstract Full Text Full Text PDF PubMed Scopus (1071) Google Scholar), InaD (42Xu X.Z. Choudhury A., Li, X. Montell C. J. Cell Biol. 1998; 142: 545-555Crossref PubMed Scopus (195) Google Scholar), and NHERF (43Maudsley S. Zamah A.M. Rahman N. Blitzer J.T. Luttrell L.M. Lefkowitz R.J. Hall R.A. Mol. Cell. Biol. 2000; 20: 8352-8363Crossref PubMed Scopus (180) Google Scholar, 44Fouassier L. Yun C.C. Fitz J.G. Doctor R.B. J. Biol. Chem. 2000; 275: 25039-25045Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). PDZ domains are also reported to interact with other distinct protein-binding motifs, including ankyrin repeats (45Maekawa K. Imagawa N. Naito A. Harada S. Yoshie O. Takagi S. Biochem. J. 1999; 337: 179-184Crossref PubMed Scopus (58) Google Scholar), spectrin repeats (46Xia H. Winokur S.T. Kuo W.L. Altherr M.R. Bredt D.S. J. Cell Biol. 1997; 139: 507-515Crossref PubMed Scopus (182) Google Scholar), and LIM domains (47Cuppen E. Gerrits H. Pepers B. Wieringa B. Hendriks W. Mol. Biol. Cell. 1998; 9: 671-683Crossref PubMed Scopus (126) Google Scholar). The structural bases for these interactions are not yet clear. A fascinating feature of PDZ-containing proteins is that they often contain multiple PDZ domains (up to 13 in the MUPP1 protein (48Ullmer C. Schmuck K. Figge A. Lubbert H. FEBS Lett. 1998; 424: 63-68Crossref PubMed Scopus (148) Google Scholar)) (Fig. 2). In many cases, the PDZ domains are closely grouped into tandem arrays, including pairs (e.g. PDZ1,2 of PSD-95) and triplets (e.g.PDZ1–3 and PDZ4–6 of GRIP). The significance of PDZ grouping is not known. However, there is some evidence to suggest that multiple domains can cooperate to enhance binding to target ligands. For instance, syntenin contains two PDZ domains in tandem. PDZ2 of syntenin binds to the C terminus of syndecan, neurexin, and ephrin-B1 only when paired with PDZ1 (or another copy of PDZ2) but does not interact when presented in isolation (49Grootjans J.J. Reekmans G. Ceulemans H. David G. J. Biol. Chem. 2000; 275: 19933-19941Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). In addition, a recent report suggests that one PDZ domain may influence the folding of an adjacent PDZ domain (50Zhang Q. Fan J-S. Zhang M. J. Biol. Chem. 2001; 276: 43216-43220Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). In this example, PDZ5 of GRIP alone was unstructured in solution by nuclear magnetic resonance and circular dichroism spectroscopy and failed to bind GluR2. However, when covalently connected to PDZ4, PDZ5 became highly structured and GluR2 binding was restored. PDZ domains are also often found in proteins with other known interaction domains or signaling domains (Fig. 2). The superfamily of proteins called membrane-associated guanylate kinases (MAGUKs), which includes PSD-95/SAP90, Dlg, and ZO-1, is characterized by one or more PDZ domains, an SH3 domain, and a catalytically inactive guanylate kinase-like domain. PDZ domains also occur in proteins with WW, LIM, and calcium/calmodulin-dependent protein kinase-like domains as well as ankyrin and leucine-rich repeats. Recently a cytoplasmic protein, PDZ-RGS3, was identified that binds B-ephrins through its PDZ domain and has a regulator of heterotrimeric G protein signaling (RGS) domain (51Lu Q. Sun E.E. Klein R.S. Flanagan J.G. Cell. 2001; 105: 69-79Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar). The multidomain structure of PDZ-containing proteins enables them to interact with multiple binding partners simultaneously, thereby assembling larger protein complexes (recently reviewed in Refs. 52Sheng M. Sala C. Annu. Rev. Neurosci. 2001; 24: 1-29Crossref PubMed Scopus (1029) Google Scholar and53Harris B.Z. Lim W.A. J. Cell Sci. 2001; 114: 3219-3231Crossref PubMed Google Scholar). PDZ-based complexes are often localized to specific subcellular compartments. PDZ-based scaffolds have been shown to organize signal transduction pathways such as phototransduction inDrosophila, where ion channels and signaling molecules are co-assembled by the multi-PDZ protein InaD (54Montell C. Curr. Opin. Neurobiol. 1998; 8: 389-397Crossref PubMed Scopus (87) Google Scholar, 55Montell C. Annu. Rev. Cell Dev. Biol. 1999; 15: 231-268Crossref PubMed Scopus (248) Google Scholar, 56Scott K. Zuker C. Curr. Opin. Neurobiol. 1998; 8: 383-388Crossref PubMed Scopus (63) Google Scholar, 57Tsunoda S. Zuker C.S. Cell Calcium. 1999; 26: 165-171Crossref PubMed Scopus (81) Google Scholar). MAGUKs appear to play a similar role in the postsynaptic density, a specialized structure at excitatory synapses enriched in glutamate receptors and associated signaling proteins (58Garner C.C. Nash J. Huganir R.L. Trends Cell Biol. 2000; 10: 274-280Abstract Full Text Full Text PDF PubMed Scopus (477) Google Scholar, 59Sheng M. Pak D.T. Annu. Rev. Physiol. 2000; 62: 755-778Crossref PubMed Scopus (298) Google Scholar, 60Scannevin R.H. Huganir R.L. Nat. Rev. Neurosci. 2000; 1: 133-141Crossref PubMed Scopus (386) Google Scholar). In both the examples of InaD and MAGUKs, the ability of PDZ-containing proteins to interact with multiple binding partners creates a protein complex specialized for local signaling functions. PDZ proteins have also been implicated in the establishment of cell polarity. In Drosophila, two PDZ-containing proteins, Bazooka and PAR-6, form a ternary complex with atypical protein kinase C that is required for proper establishment and maintenance of apical-basal polarity in epithelial tissues (reviewed in Ref. 61Ohno S. Curr. Opin. Cell Biol. 2001; 13: 641-648Crossref PubMed Scopus (371) Google Scholar). The orthologues of these binding partners in C. elegans are likewise necessary for polarization of the one-cell embryo. Importantly, depletion of any one of the proteins results in mislocalization of the other two, underscoring the functional importance of the complex. The LIN-2·LIN-7·LIN-10 tripartite complex also demonstrates a role for PDZ proteins in protein sorting (62Kim S.K. Curr. Opin. Cell Biol. 1997; 9: 853-859Crossref PubMed Scopus (104) Google Scholar, 63Bredt D.S. Cell. 1998; 94: 691-694Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). This complex specifies the basolateral targeting of the epidermal growth factor receptor homolog LET-23 in C. elegans, and the mammalian homologs have been proposed to play a role in NMDA receptor trafficking through an interaction with the kinesin superfamily motor protein KIF17 (64Setou M. Nakagawa T. Seog D.H. Hirokawa N. Science. 2000; 288: 1796-1802Crossref PubMed Scopus (597) Google Scholar). These examples and others illustrate the role of PDZ-containing proteins in determining the subcellular location of their binding partners. Two recent studies also indicate that PDZ domains can modulate the function of their associated proteins as well as the localization, as interaction of the CFTR with either the multi-PDZ adaptor protein CAP70 or NHERF increases chloride channel activity of the CFTR protein, perhaps by promoting multimerization of the channel protein (65Wang S. Yue H. Derin R.B. Guggino W.B. Li M. Cell. 2000; 103: 169-179Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar, 66Raghuram V. Mak D.D. Foskett J.K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1300-1305Crossref PubMed Scopus (195) Google Scholar). Thus PDZ domains may have a functional role in modulating the activity of their target ion channels and membrane receptors (67Bezprozvanny I. Maximov A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 787-789Crossref PubMed Scopus (80) Google Scholar). It is now clear that PDZ domain proteins play an important role in the targeting of proteins to specific membrane compartments and their assembly into supramolecular complexes. There is also evidence that they can regulate the function of their ligands in addition to serving as scaffolds. Their ability to bind short extreme C-terminal sequences offers a facile way for PDZ proteins to interact with target proteins without disrupting the overall structure and function of their ligands. Key questions remain in understanding how PDZ domains serve their function. For example, most PDZ domains seem to bind multiple ligands. What determines the interaction with specific binding partners and how is this regulated spatially and temporally in the cell? How is ligand binding and protein stoichiometry affected by PDZ-based homo- and heteromultimerization, and what is the structural basis for multimer formation? What role does the tandem organization of PDZ domains play in determining scaffolding function? The answers to these questions and others await investigation in this exciting and evolving field.