REALIDAD AUMENTADA

The cloning o f the first glutamate receptor subunit in 1989 was a landmark event in the study o f these receptors. Due to the difficulties in purifying the glutamate receptors, partial protein sequences could not be obtained. Therefore, a new technique, expression cloning was used (see 1.3.3.1). Utilizing this technique to search a rat brain cDNA library the first glutamate receptor clone, GluRl was isolated (Hollmann et al, 1989). Using cDNA probes derived from this sequence other related clones were isolated by homology screening and PGR. Molecular cloning has now identified 9 genes encoding subunits o f non-NMDA glutamate receptors, GluRl-GluR7 and KA-1 and KA-2 (reviewed in Hollmann and Heinemann, 1994). The AMPA receptor subunits are GluRl-G luR4. The kainate receptor subunits are KA-1 and KA-2, which generate the high affinity [^H]kainate binding sites and GluR5-GluR7 which are responsible for the low affinity [^H]kainate binding sites (Bettler and Mulle,

1995).

G luR l-G luR 4 clones

The GluRl cDNA has an open reading frame o f 2.7 kb and encodes a mature polypeptide sequence o f 889 amino acids with a calculated M^ o f 99769. This was the largest ligand-gated ion channel subunit then reported, however, the NR2 subunits are now known to be considerably larger (see 1.3.3.1). Hydropathy analysis o f the sequence predicted four hydrophobic regions o f sufficient length to form transmembrane a-helices. Protein sequence comparison with members o f the ligand-gated ion channel superfamily, such as nAChR and GABA^R subunits revealed an overall amino acid homology of only -2 0%, despite apparent structural homology. The further three clones encoding putative AMPA receptor subunits, GluR2-GluR4, were similar to G luR l. The GluR2-GluR4 cDNAs encoded protein sequences o f 862, 8 6 6 and 881 amino acids, with predicated

molecular mass o f 96400, 98000 and 101034 Da respectively. The overall amino acid sequence identity o f GluRl-G luR4 was 70%, see Figure 1.8. These subunits are highly conserved between species, the rat, mouse and human GluRl subunits have 96-97% amino acid identity. Each o f the G luRl-G luR4 subunits has two splice forms, due to alternative splicing o f an exon immediately prior the M4 region (Figure 1.8). The alternative exons have been termed ’flip' and 'flop' (reviewed in Hollmann and Heinemann, 1994). A further splice variant for the GluR4 subunit, GluR4c, which is due to an alternative C-terminus o f 38 amino acids has been described (Gallo et a l, 1992). The alternative C-terminus can exist with either the 'flip' or 'flop' form. A further mechanism for creating diversity is RNA editing. The genes for GluRl-GluR4 all encode a glutamine (Q) residue in the putative M2 region (Figure 1.8). However, for all the GluR2 cDNA clones from adult animals analysed, the glutamine residue at position 582 was replaced by an arginine (R) (Sommer et ai, 1991). The selective RNA editing is due to a specific intron sequence in the GluR2 gene (Egebjerg et al,

1994). A proposed mechanism is; a dsRNA structure forms creating a substrate for a dsRNA-specific adenosine deaminase. This enzyme could convert the adenosine to inosine, which could serve as a template for the incorporation of cytosine by reverse transcriptase. Thus the codon would be changed from CAA to GAG i.e. Q to R (Rueter et a l, 1995).

GluR5-GluR7 clones

Using low-stringency hybridization screening with probes derived from the G luR l-G luR 4 clones and PGR amplification with degenerate primers the first o f the kainate receptor subunit cDNAs, GluR5 was cloned (Bettler et a l, 1990). This was followed by the cloning o f the GluR6 and GluR7 subunit cDNAs (Egebjerg et a l, 1991; Bettler et a l, 1992). The cDNAs encode for proteins of approximately 900 amino acids and ~ 100000. The GluR5, GluR6 and GluR7 subunits share 70-80% amino acid identity with each other, but only ~ 40% with the G luRl-G luR4 subunits. As with the GluRl-GluR4 subunits, 4 membrane

Figure 1.8 A comparison o f the polypeptide sequences encoded by the rat AMPA receptor cDNAs, GIuRl-GluR4

Identical amino acids are represented by (*), conservative substitutions are indicated by (.). The putative membrane regions, M1-M4, are shown by (h )- The

flip/flop segment is shown by ( --- ). The signal peptide and Q/R site are indicated. The sequence alignment was performed by PC/gene software package, using the method o f Higgins and Sharpe (1988).

leading peptide GluRl M P Y I F A F F C T G F L G A W G GluR2 M-QKIMHISVLLSPVLWGLIFG GluR3 MGQSVLRAVFFLVLGLLGHSHG GluR4 M-RIICRQIVLLFSGFWGLAMG ANFPNNIQIGGLFPNQQSQEHAAFRFALSQLTE 33 V-SSNSIQIGGLFPRGADQEYSAFRVGMVQFST 32 G-FPNTISIGGLFMRNTVQEHSAFRFAVQLYNT 3 2 A-FPSSVQIGGLFIRNTDQEYTAFRLAIFLHNT 32 * * * * ilr ★ ★ "k "k 'k GluRl PP--- KLLPQIDIVNISDTFEMTYRFCSQFSKGVYAIFGFYERRTVNMLTS 81 GluR2 SE--- FRLTPHIDNLEVANSFAVTNAFCSQFSRGVYAIFGFYDKKSVNTITS 81 GluR3 NQNTTEKPFHLNYHVDHLDSSNSFSVTNAFCSQFSRGVYAIFGFYDQMSMNTLTS 87 GluR4 SPNASEAPFNLVPHVDNIETANSFAVTNAFCSQYSRGVFAIFGLYDKRSVHTLTS 87 •k k k k k k k k k k k k k k k k k k GluRl FCGALHVCFITPSFPVDTSNQFVLQLRPELQEALISIIDHYKWQTFVYIYDADRG 136 GluR2 FCGTLHVSFITPSFPTDGTHPFVIQMRPDLKGALLSLIEYYQWDKFAYLYDSDRG 136 GluR3 FCGALHTSFVTPSFPTDADVQFVIQMRPALKGAILSLLSYYKWEKFVYLYDTERG 142 GluR4 FCSALHISLITPSFPTEGESQFVLQLRPSLRGALLSLLDHYEWNCFVFLYDTDRG 142 GluRl LSVLQRVLDTAAEKNWQVTAVNILTTTEE G Y R M L F Q D L E K K K E R L V W D C E 187 GluR2 LSTLQAVLDSAAEKKWQVTAINVGNINNDKKDETYRSLFQDLELKKERRVILDCE 191 GluR3 FSVLQAIMEAAVQNNWQVTARSVGNIKD VQEFRRIIEEMDRRQEKRYLIDCE 194 GluR4 YSILQAIMEKAGQNGWHVSAICVENFND V-SYRQLLEELDRRQEKKFVIDCE 193

k k k k k k k k k k k k GluRl SERLNAILGQIVKLEKNGIGYHYILANLGFMDIDLNKFKESGRNVTGFQLVNYTD 242 GluR2 RDKVNDIVDQVITIGKHVKGYHYIIANLGFTDGDLLKIQFGGANVSGFQIVDYDD 246 GluR3 VERINTILEQWILGKHSRGY H Y M L A N L G F T D I L L E R V M H G G A N I T G F Q I V N N E N 249 GluR4 lERLQNILEQIVSVGKHVKGYHYIIANLGFKDISLERFIHGGANVTGFQLVDFNT 248 * * * * * * * * * * * * * * * * * * * * GluRl TIPARIMQQWRTSDSRDHTRVDWKRPKYTSALTYDGVKVMAEAFQSLRRQRIDIS 2 97 GluR2 SLVSKFIERWSTLEEKEYPGAHTATIKYTSALTYDAVQVMTEAFRNLRKQRIEIS 301 GluR3 PMVQQFIQRWVRLDEREFPEAKNAPLKYTSALTHDAILVIAEAFRYLRRQRVDVS 304 GluR4 PMVTKLMDRWKKLDQREYPGSETPP-KYTSALTYDGVLVMAETFRSLRRQKIDIS 302 k k k k k k k k k k k k k k k k GluRl RRGNAGDCLANPAVPWGQGIDIQRALQQVRFEGLTGNVQFNEKGRRTNYTLHVIE 352 GluR2 RRGNAGDCLANPAVPWGQGVEIERALKQVQVEGLSGNIKFDQNGKRINYTINIME 356 GluR3 RRGSAGDCLANPAVPWSQGIDIERALKMVQVQGMTGNIQFDTYGRRTNYTIDVYE 359 GluR4 RRGNAGDCLANPAAPWGQGIDMERTLKQVRIQGLTGNVQFDHYGRRVNYTMDVFE 357 k k k k k k k k k k k k k k k k k k k k k k * k ^ k k k k k GluRl MKHDGIRKIGYWNEDDKFVPAATDAQAGGDNSSVQNRTYIVTTILEDPYVMLKKN 407 GluR2 LKTNGPRKIGYWSEVDEOyrWTLTELPSGNDTSGLENKTVWTTILESPYVMMKKN 411 GluR3 MKVSGSRKAGYWNEYER F V P F S D Q Q - I S N D S S S S E N R T I W T T I L E S P Y V M Y K K N 413 GluR4 LKSTGPRKVGYWNDMDKLVLIQD M P T L G N D T A A I E N R T V W T T I M E S P Y V M Y K K N 412 k k k k k k k k k k k k k k k * * * * * * * * GluRl ANQFEGNDRYEGYCVELAAEIAKHVGYSYRLEIVSDGKYGARDPDTÏCAWNGMVGE 462 GluR2 HEMLEGNERYEGYCVDLAAEIAKHCGFKYKLTIVGDGKYGARDADTKIWNGMVGE 466 GluR3 HEQLEGNERYEGYCVDLAYEIAKHVRIKYKLSIVGDGKYGARDPETKIWNGMVGE 468 GluR4 HEMFEGNDKYEGYCVDLASEIAKHIGIKYKIAIVPDGKYGARDADTKIWNGMVGE 467 _*** ****** _** ***** ,*...*■*■ *****•*■**,,** ******* GluRl LVYGRADVAVAPLTITLVREEVIDFSKPFMSLGISIMIKKPQKSKPGVFSFLDPL 517 GluR2 LVYGKADIAIAPLTITLVREEVIDFSKPFMSLGISIMIKKPQKSKPGVFSFLDPL 521 GluR3 LVYGRADIAVAPLTITLVREEVIDFSKPFMSLGISIMIKKPQKSKPGVFSFLDPL 523 GluR4 LVYGKAEIAIAPLTITLVREEVIDFSKPFMSLGISIMIKKPQKSKPGVFSFLDPL 522 * * * * * _ * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * Ml GluRl A Y E I W M C I V F A Y I G V S W L F L V S R F S P Y E W H S E E F E E G R D — QTTSDQSNEFGIF 570 GluR2 A Y E I W M C I V F A Y I G V S W L F L V S R F S P Y E W H T E E F E D G R E T Q — SSESTNEFGIF 57 4 GluR3 AYEIWMCIV F A Y I G V S W L F L V S R F S P Y E W H L E D N N E E P R D P Q S P P D P P N E F G I F 578 GluR4 A Y E I W M C I V F A Y I G V S W L F L V S R F S P Y E W H T E E P E D G K E G P — SDQPPNEFGIF 575 * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * *

M2 M3 GluRl NSLWFSLGAFMQQGCDISPRSLSGRIVGGVWWFFTLIIISSYTANLAAFLTVERM 625 GluR2 NSLWFSLGAFMRQGCDISPRSLSGRIVGGVWWFFTLIIISSYTANLAAFLTVERM 629 GluR3 NSLWFSLGAFMQQGCDISPRSLSGRIVGGVWWFFTLIIISSYTANLAAFLTVERM 633 GluR4 NSLWFSLGAFMQQGCDISPRSLSGRIVGGVWWFFTLIIISSYTANLAAFLTVERM 630 * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * Î Q/R site GluRl VSPIESAEDLAKQTEIAYGTLEAGSTKEFFRRSKIAVFEKMWTYMKSAEPSVFVL 680 GluR2 VSPIESAEDLSKQTEIAYGTLDSGSTKEFFRRSKIAVFDKMWTYMRSAEPSVFVR 684 GluR3 VSPIESAEDLAKQTEIAYGTLDSGSTKEFFRRSKIAVYEKMWSYMKSAEPSVFTK 688 GluR4 VSPIESAEDLAKQTEIAYGTLDSGSTKEFFRRSKIAVYEKMWTYMRSAEPSVFTR 685 ' k ' k - k ' k ' k ' k ' k ' i e ' k ' k - k ' k ' k ' k - k ' k ' k ' k ' k - k ' k ' k - k ' k * * -A: ★ ★ * * i&r * * * GluRl TTEEGMIRVRKTKGKYAYLLESTMNEYIEQRKPCDTMKVGGNLDSKGYGIATPKG 735 GluR2 TTAEGVARVRKSKGKYAYLLESTMNEYIEQRKPCDTMKVGGNLDSKGYGIATPKG 739 GluR3 TTADGVARVRKSKGKFAFLLESTMNEYIEQRKPCDTMKVGGNLDSKGYGVATPKG 7 43 GluR4 TTAEGVARVRKSKGKFAFLLESTMNEYTEQRKPCDTMKVGGNLDSKGYGVATPKG 740 * * _ _ * _ * * * * _ * * * _ * _ * * * * * * * * * _ * * * * * * * * * * * * * * * * * * * * * * * * * *

"flip or flop" segment

GluRl SALRNPVNLAVLKLNEQGLLDKLKNKWWYDKGECGTGGGDSKDKTSALSLSNVAG 7 90 GluR2 SSLGNAVNLAVLKLNEQGLLDKLKNKWWYDKGECGSGGGDSKEKTSALSLSNVAG 7 94 GluR3 SALGNAVNLAVLKLNEQGLLDKLKNKWWYDKGECGSGGGDSKDKTSALSLSNVAG 7 98 GluR4 SSLGNAVNLAVLKLNEQGLLDKLKNKWWYDKGECGSGGGDSKDKTSALSLSNVAG 7 95 * * * • k - k - k ' k - k - k ' k - k ' k ' k ' k ' k ' k ' k ' k ' k - i e ' k ' k ' k - k - k ' k ' k ' k ' k - k ' k ' k -k -k -k -k -k ' k k - k k - k k k k k k k - k k M4 GluRl VFYILIGGLGLAMLVALIEFCYKSRSESKRMKGFCLIPQQSINEAIRTSTLPRNS 845 GluR2 VFYILVGGLGLAMLVALIEFCYKSRAEAKRMK--- 82 6 GluR3 VFYILVGGLGLAMMVALIEFCYKSRAESKRMK--- L T --- 832 GluR4 VFYILVGGLGLAMLVALIEFCYKSRAEAKRMK--- LTFSEATRNK--- 837

* * * * * * * * * * * * * * * * * * * * * * * _ * ^ * * * *

GluRl GAGASGGG G S G E N G R W S Q D F P K S M Q S I P S M S H S S G M - P L G A T G L 88 9 GluRl GluR2 --- VAKNPQNINPSSSQNSQNFATYKEGYNVYGIESVKI 862 GluR2 GluR3 --- KNTQNFKPAPATNTQNYATYREGYNVYGTESVKI 8 66 GluR3 GluR4 -ARLSITGSVGENGRVLTPDCPKAVHTGTAIRQSSGLAVIASDLP 881 GluR4

regions are proposed. Both the GluR5 and GluR6 clones are RNA edited at a homologous position to the GluR2 cDNA, although not completely, approximately 39% o f GluR5 cDNA and 75% o f GluR6 cDNA was found to be in the edited (R) form. The GluR5 clone also has several splice variants, due to the presence or absence o f an exon in the N-terminal domain and in the presence or absence o f exons in the C-terminus. The N-terminal and C-terminal splice variations can occur together and can be RNA edited or not (reviewed in Hollmann and Heinemann, 1994).

KA-1 and KA-2 clones

Two high affinity kainate receptor subunits have been cloned, KA-1 and KA-2, the cDNAs encoded for polypeptide sequences o f 936 and 965 amino acids respectively. They share the same overall structure as the GluRl-GluR7 subunits. However, while KA-1 and KA-2 share 70% amino acid identity, with the GluR5-GluR7 subunits with the GluRl-GluR4 subunits they have only ~ 43% and - 37% amino acid identity respectively. Splice variants have not been reported for KA-1 or KA-2, nor is there any indication to date o f RNA editing. Both subunits contain a Q at the homologous M2 site (reviewed in Hollmann and Heinemann, 1994).

Subunit topology

The original topology o f the GluR subunits was proposed to be four transmembrane regions, due to potential similarity to the nAChR, with the N- terminus extracellular and the C-terminus intracellular, see Figure 1.4 Model 1. However, as for the NMDA receptor subunits (see 1.3.3.1) this proposed topology increasingly appears incorrect. Studies suggest that the domain between the M3 and M4 region is extracellular. In the GluR6 subunit, a consensus glycosylation site was found to be glycosylated in vivo (Roche et al., 1994). Hollmann et al (1994) also demonstrated A-glycosylation of the same domain in the GluRl subunit when expressed in Xenopus oocytes. The extracellular location o f the M3-M4 domain implied an intracellular C-terminal domain. This

was further supported by the immunocytochemical studies on the GluRl subunit (Martin et a l, 1993; Molnar et a l, 1993). Wo and Oswald (1994) demonstrated that the removal o f the M2 region did not alter the topology o f the mutated kainate receptor subunit as indicated by A/^-glycosylation. Thus the M2 region did not span the membrane. Bennett and Dingledine (1995) used an epitope protection assay to investigate the topology o f the GluR3 subunit. This technique demonstrated an intracellular location for the C-terminus, the extracellular location o f the flip/flop region and that both ends o f the proposed M2 region were intracellular. The resulting topological model for the glutamate subunits from these studies and studies carried out on the NMDA receptor subunits (see 1.3.3.1) is shown in Figure 1.4, model 2.