Receptor classes and the transmitter-gated ion channels

Signalling at the plasma membrane

THE TRANSMITTER-GATED ion channels form a class of multisubunit membrane-spanning receptors that are essential for rapid signal transduction. The property that defines this class is that the transmitter molecule itself operates the opening or closing of the channel by binding to a site on the receptor. These channels are but one of several classes of the receptor now known to act in membrane signal transduction (Table I). This classification covers the many hundreds of receptor types which are concerned with signal transmission between neurones, between glia and neurones or between neurones and muscles or other effector organs, as well as with the responses of glands and muscles to circulating signals. The other receptors, of quite different functions, which are involved primarily in regulation of gene expression (see the articles by P. Chambon and by M. Karin, this issue) or in the transport of metabolites, transmitters and so on into ceils, as well as those membrane receptors specific to the immune system (see article by R. Abraham, this issue) are excluded.

Membrane receptor classes Molecular cloning has recently yielded the amino acid sequences of many (although far from all) of the subunits of the signal-transducing membrane receptors. These can then be divided into three classes based on their subunit structure. It is important to note that, unusually, it is the secondary structure of the subunits, rather than primary structure homology, that determines the receptor class. Different receptors may be monomeric or polymeric (homomeric or heteromeric), but all of the subunits contain at least one hydrophobic, transmembrane domain (TM). The number and arrangement of these TMs is a major factor in defining the classes of receptors (Table I). Class 1 receptor subunits have structures that can assemble in an oligomer surrounding a membrane pore. Transduction in this case is via the opening of a cation or an anion channel [or, in one case (Table liB), the closing of a cation channel].

E. A. Barnard is at the MRC Molecular NeurobiologyUnit, Medical Research Council Centre, Hills Road, Cambridge, UK CB2 2QH.

368

TIBS 17 - OCTOBER 1992

Receptor classes and the transmitter-gated ion channels

Transmitter-gated channels, which can be selective for cations or for anions, form an important class among the membrane receptors responsible for signal transduction. Thirteen principal types of these channels can now be recognized and most of these are available for analysis in recombinant form. It is instructive to contrast their characteristic structural features with those of the two other primary classes of the signaltransducing receptors of membranes. In Class 2, most of the subunit mass neurotrophin receptors, there is a secis packed within the membrane and ond subunit (containing a peptide each subunit carries a G protein recog- single-TM domain) present in the renition sequence on its intracellular face. ceptor, to confer high-affinity ligand Although most members of this class binding and a transduction element, i.e. share some fractional sequence identity, those receptors in one subset are heteroit is in fact only the seven-span TM dimers and the product of two genes 1,2. structure which is common to all of them, since there are at ]east two smaller Transmitter-gated ion channels These receptors perform fast sigsets which exhibit no sequence homology whatsoever to the others in this nalling, since their transduction is independent of any intracellular or memclass (Table I). The Class 3 subunit structure pro- brane-diffusible factor. In addition to vides a minimum exposure of the sub- the usual case of receptor activation by unit to the membrane lipids (with only a presynaptically released transmitter one traversal of the bilayer), which (Class 1A, Table II), the transmitter facilitates receptor mobility and intern- molecule may arrive on the intracellular alization. It can be noted that all of the side (Class 1B, Table If). The latter subreceptors in that class produce, as one division includes those receptors part of their signalling function, long- where the signalling occurs across an term, nuclear-based effects. These re- organelle membrane {the ryanodine ceptors can now be divided into two receptor and most of the inositol 1,4,5very distinct structural subclasses trisphosphate [Ins(1,4,5)P3] receptors}, (Table I). Class 3.l contains several in which Ca 2+ ions are transferred from superfamilies, which again share no an intracellular store in response to the sequence homology except within binding of Ins(1,4,5)P3 or, for the ryanthemselves, but all possess one TM odine receptor 3, some other signal, perinternal peptide sequence. Class 3.II haps Ca2+itself. Most structural and functional inforcontains only one member so far, the CNTF receptor, but some other growth mation is available for the nicotinic factor receptors might be found to be acetylcholine (ACh) receptor of skeletal similarly constructed. In the ligand- muscles or electric organs 4 and in this binding subunit of the CNTF receptor, the case it has been definitively established single membrane insertion consists of a that all of the subunits span the memcarboxy-terminal glycosylphosphatidyl- brane, forming a pentamer which eninositol (GPI) linkage 1. All of the poly- closes a central ion channel 5. There are peptide chain is extracellular, so there four subunit types, ~, J3, T and 8, which is no intracellular transduction domain. are in the stoichiometry c%13¥8. It is This structure could facilitate the re- interesting that a cation channel within lease of a diffusible form of the receptor neuronal nicotinic receptors 4 has eswhen required: a similar principle is sentially the same properties as the used in membrane-bound forms of above, except that it is formed from acetylcholinesterase. In 3.II, however, only two subunit types, o¢ and 13. Here as in some of the Class 3.I cytokine and again the structure is pentameric (~2133, © 1992,ElsevierSciencePublishers, (UK) 0376-5067/92/$05.00

Signalling at

TIBS 1 7 - OCTOBER 1992

the

plasma m e m b r a n e

Table I. Signal-transducing receptors of membranes: structural classification Class

Subunit composition

Transduction system

Ligands

1. Channel-enclosing oligomers (A) Extracellularly activated (B) Intracellularly activated

Heteromeric or homomeric

Transmitter-gated ion channels

See Table II

2. Seven-hydrophobic-domain polypeptides Super-familiesC: I Main superfamily II Secretin, VIP, parathyroid hormone and calcitonin receptors 46 III Metabotropic glutamate receptors 47

Monomers or homodimers or post-translational heterodimers a

Via a G protein (A) Plus a diffusable messenger

3. Single-hydrophobic-domain Monomers or homodimers polypeptides or post-translational I Containing one TM sequence heterotetramersg or native (in several superfamilies) heterodimers h or II Containing no TM, but has heterotrimers i a glycolipid membrane anchor

(A) All the small transmitters (except glycine); neuropeptides; odorants; certain cytokines (e.g. IL-8); lipid and related agonists (PAF, eicosanoids) (B) Acting directly on a channeP (B) Atrial muscarinic; neuronal C~l-adrenergic,etc. (C) After receptor cleavage by (C) Thrombin is the only case so far known48 a polypeptide hormone acting as a site-specific protease (and not as a classical agonist) to form a self-activating receptor d The binding subunit itself is: (A) a ligand-stimulated tyrosine kinase (B) a ligand-stimulated guanylate cyclase49 (C) not of known enzymatic activity

All are polypeptides: (A) mitogenic growth factorse; insulin (B) natriuretic peptides49 (C) neurotrophins; growth hormone, prolactin and many cytokines f. Of type II, only CNTF-R is so far known

aln this class only the thyrotropin receptor is known, so far, to contain two types of extraceliular chain, and these arise post-translationally from one poiypeptide precursor 1. bReviewed in Ref. 50. teach has no sequence homology to the others. din at least one type of thrombin receptor (which is in superfamily I), thrombin specifically cleaves the receptor in its extracellular domain to liberate a new amino-terminal segment therein: this leads to self-activation of the receptor and hence intracellular signalling events 48. eSee M. J. Pazin and L. T. Williams, this issue, fSee A. Miyajima, this issue, glnsulin and insulin-like-growth-factor I receptors, hArt independent second subunit may confer high-afinity binding and tyrosine kinase activity (neurotrophin receptors) or some other transduction (CNTF-R, some cytokine receptors) 1'2. iThe IL-2 receptor 51. Abbreviations: TM, transmembrane domain; CNTF-R, ciliary neurotrophic factor receptor; IL, interleukin; PAF, platelet activating factor; VIP, vasoactive intestinal polypeptide.

at least in the cases analysed) 6. There is evidence that the glycine receptor also has the pentameric structure 7, although forming an anion channel, and electron optical studies have recently indicated a further pentameric structure for the GABAA receptors (N. Nayeem, G. Zampighi, N. Unwin and E. A. Barnard, unpublished). All eukaryotic channel proteins are characterized by highly hydrophobic stretches of 19-27 amino acid residues. Apart from the amino-terminal signal peptide regions (which are removed after translation), these segments are generally regarded as the transmembrane domains, since they can span the bilayer if in a perpendicular or (for the longer lengths) tilted helix conformation. The subunits of the nicotinic ACh, GABAA, glycine, 5-HT3 and (so far as they are known) the glutamate receptors each contain four such TMs in the carboxy-terminal region of the sequence (Fig. 1). However, the TMs differ significantly in length even within one polypeptide, and it cannot yet be assumed that they are all (z-helical;

other transmembrane structures may be possible for some of them. The nicotinic ACh, GABA and glycine receptor subunits have precisely the same distribution of these TMs along the chain. This, together with the partial sequence homologies observed over almost the whole length of the three subunit types, led to the proposal of a superfamily of these transmitter-gated channels 9. This must now be assigned as superfamily Ia (Table II); some other subunits in the Class 1 extracellularly activated channels differ very greatly in sequence between themselves (in the glutamate receptors, discussed below) but still have four TMs at fairly similar relative spacings. These can therefore be designated superfamily Ib. In addition, the intracellularly activated channels fall into two other superfamilies, II and III, by their sequences and their differing distributions of TMs (Table IF). For the lns(1,4,5)P3 and ryanodine receptors the hydrophobicity profiles are more complex3,'°. Mikoshiba and colleagues" have recently deduced, using both immunogold label-

ling and species sequence comparison, that there are six TMs in the subunit of the Ins(1,4,5)P receptor, which (like the ryanodine receptor) is homotetrameric. These two receptors display good sequence homology over two of the TMs, but much less elsewhere. The cGMP-gated and (cAMP- and cGMP)gated receptors are 57% identical in sequence ~2, but exhibit no homology at all with the Ins(1,4,5)P3/ryanodine receptor type and neither set has any homology with the superfamilies Ia or Ib. The assignment 12,~3of six TMs to the cyclic nucleotide-gated channels in Table If refers only to the apparently t~helical hydrophobic sequences present in their subunits. These subunits exhibit some sequence and structural similarities to several types of voltage-gated cation channels and the pattern of their TMs has been deduced to correspond to some in the latter channels TM. There is also particular homology in an 'H5' region, lying between the fifth and sixth hydrophobic TMs in both cases. There is evidence that this H5 or 'SS1-SS2' stretch (made up of ~20 hydrophobic

369

Signalling at the plasma membrane Table II. Transmitter-gated ion channels Operator ligand

Ion Superfamily selectivitya

Transmembrane References domains

A. Extracellularly activated GABAA Glycine ACh (nicotinic, muscle type) ACh (nicotinic, neuronal) Glutamate : non-NMDA Glutamate : NMDA 5-HT3 ATP (P2x,channel-opening)

CI-, HCO~ CI-, HCO3 Na+,K+,Ca2+ Na+,K+,Ca2÷ Na+,K+,(Ca2+) Na+,K+,Ca2÷ Na+,K+ Ca2+,Na+,Mg2+

la la la la tb Ib ta ?

4 4 4 4 4 4 4

9 7 4 4 31 38 19 52

Na+,K+ Na+,K+ K÷ Ca2+

II II ? III

(6) (6) (6)

13 12 53 10,11

Ca2+

III

(6)

3

B. Intracellularly activated cGMP (photoreceptors) cAMP, cGMP (olfactory neurones) ATP (channel-closing) Ins(1,4,5)P3 (organelles and plasma membrane) (?)Ca2+ (ryanodine receptor)

This table lists all of the receptors of the nervous system so far known in which an endogenous signalling molecule activates the opening or closing of an ion channel contained in the receptor structure. Where their protein sequences are known, they fall into the entirely distinct superfamilies (I, II and Ill) of homologous proteins indicated. Superfamilies la and Ib show only a very low degree of sequence similarity. The transmembrane domains are inferred from a hydropathy plot of the protein sequence: in some cases the assignment is less clear, indicated by the numbers in parentheses. aFo[ the Ca 2+ permeabilities in group A, see the section on glutamate receptors. The muscle ACh receptor Ca 2+ flux is low, but in the neuronal type it is high (Table IV) 54'55. Parentheses: significant in a minority of types only31. Abbreviations: GABA, y-aminobutyrate; ACh, acetylcholine; NMDA, /kLmethyl-D-aspartate; 5-HT, serotonin; Ins(1,4,5)P3, inositol trisphosphate; P2x, purinergic receptor, type 2x.

and hydroxy amino acids and prolines) is in the conduction pore in the voltagegated Na+ and K+ channels, where it forms a [3-hairpin structure which enters the membrane is. On this model there may, therefore, be a seventh (nonhelical) transmembrane domain, which is the channel-lining domain, in the cyclic nucleotide-gated subclass of receptor channels.

The receptors of superfamily la The

GABA A r e c e p t o r

is t h e

major

inhibitory neurotransmitter receptor of the brain and is present on the majority of its neurones in virtually all regions. Glycine likewise opens an anion channel but in the mature mammal its receptor is largely restricted to the brainstem and spinal cord 7. By contrast, the 5-HT3 and the two nicotinic ACh receptor families contain a cation channel and have somewhat restricted and very different distributions. It is striking, therefore, that all of these five receptor types are composed of subunits with some sequence homology and with the same deduced topology illustrated in Fig. 1. All five are therefore in the same superfamily. The model (Fig. 1) for their common structure shows an extracellu-

370

lar domain which extends over the entire amino-terminal half of the subunit. In all the GABA and glycine receptor subunits there is an excess of positively charged amino acid side chains towards the ends of the proposed TM domains. Clusters of Arg and Lys residues at the channel mouth are, therefore, presumed to determine entry to the anion channel, since in the nicotinic ACh and 5-HT3 receptors most of those positions are occupied instead by neutral or negatively charged residues. A general feature of interest in this superfamily is the long cytoplasmic loop, found between the TM3 and TM4 domains. In the nicotinic ACh receptor this also contains a distinct amphipathic structure (MA), i.e. a sequence which could form an co-helix with one polar and one hydrophobic face (Fig. 1). This is present in all of the ACh receptor subunits known, both from muscle and brain and including invertebrate subunits 16. 5-HT3, GABAAand glycine receptors do not possess an MA sequence, so it is not required in this superfamily for the channel structure in general, nor specifically for a cation channel. Perhaps it interacts with a cytoskeletonbridging protein for nicotinic receptors

TIBS 17 - OCTOBER 1992 (e.g. the '43K protein', for the muscle type17). A set of more than 80 subunit sequence isoforms of the subunits of the GABAA, glycine, 5-HT3, muscle nicotinic and neuronal nicotinic receptors from a very wide range of species is now available. This considerable database allows the detection of invariant sequence features, which are likely to be important. Some of these are common to the cation and the anion channel subunits and some are specific to each (Table IIl). An interesting constant feature in all subunit types is a pair of cysteines at a 15-residue spacing at a fixed position in the extracellular domain, which are known (at least in the case of the nicotinic ACh receptor) to form a disulfidebridged loop. There is a consensus sequence for this loop in all five receptor types (Table III), suggesting that it is important in determining some common features of the tertiary structure of the extracellular domain, which contains the transmitter-binding sites. This loop, as a mini-domain, is very suitable for molecular modelling in all of the subunit types of these Ia receptors, when the presence of a constant [3-folded amphipathic structure with a fixed-position aspartic acid side chain is predicted 18. TM2 is the most hydrophilic of the four TMs in all these receptor subunit types, but the TM2 sequences are quite different between the anion and cation channels, having many hydroxy side chains in the former and flanking negative charges in the latter. This is consistent with the body of evidence that TM2 lines the aqueous ion channel in the muscle-type nicotinic receptor 4 and suggests that this may be true for all members of this group. In the nicotinic receptors (electric organ, muscle and neuronal) all ~-subunits carry a pair of adjacent disulfide-bonded cysteines just before TM1, a feature associated with cholinergic ligand binding 4. This feature is absent in the non-~-subunits of the nicotinic receptors and in the anion channel receptors. However, it is not intrinsic to the activation mechanism for a cation channel in this superfamily, since transmitter binding and channel opening occur in a recombinant 5-HT3 receptor 19 where the feature is absent, despite an overall subunit homology to the nicotinic receptors.

GABAA receptor types: overwhelming numbers Since the original cloning and expression of cDNAs encoding an 0¢-and a

TIBS 17 - OCTOBER 1992

~subunit 9 and of several isoforms of ix (Ref. 20), the number of subunit types isolated by molecular cloning has greatly multiplied. Five different subunit types (with 30--40% identity between pairs) of the GABAA receptor (ix, 9, T, 15 and p) have been thus recognized, each except 8 having multiple isoforms (65-80% identity). Work from several laboratories has resulted in the reporting, to date, of the amino acid sequences for six ix-subunits (ixl-ix6), four ~-subunits ([31-[34), three ~-subunits (71-73), a 8-subunit, as well as pl and p2 in the retina. The original references to all of their sequences are given in a recent review8, updated by Cutting et al. 21 for p2, Refs 22,45 for y3 and Harvey et al. 23 for an invertebrate GABAA receptor type. Each isoform is encoded by a separate gene; all are homologous, of similar size and have the general structure shown in Fig. 1. Even in an invertebrate GABAA receptor (cloned from a mollusc) the characteristic subunit features are conserved and in co-expression experiments, a functional molluscan subunit, can replace one in the mammalian GABAA receptor 23. The co-expression of the various mammalian subunits, in either Xenopus o o c y t e s 9'20'45 o r transfected mammalian cells (e.g. Ref. 22), has allowed the exploration of some of the differing pharmacologies produced by alternative subunit combinations. Thus, the y2- or T3-subunit (but not the ~/1-subunit) confers the normal response to benzodiazepines (BZs) of both the positive and the negative modulatory types, upon a complex of co-expressed ix- and ~-subunits 8,22,45,whereas if a 8-subunit is added instead there is no BZ sensitivity. Significant functional differences have also been detected when the isoform of ix in a given ternary combination is changed, as in responses to GABA8,2°, or a neurosteroid modulator 24, or various BZ agonists (reviewed in Ref. 8); in the extreme cases of ix4 and ix6 BZ sensitivity is abolished. Such co-expression studies have led us to the view that the GABAAreceptors in vivo are a combinatorial series constructed from different isoforms of these subunits. For the BZ-sensitive types this would require, on present knowledge, one of the ixl3? combinations in a pentamer (though the stoichiometries within it are unknown). However, since BZ-insensitive types have been detected in native GABAA receptors at some locations, this would indicate that additional combinations exist

Signalling at (a)

the

plasma m e m b r a n e (b)

H2N

I

GABA

ACh c

OUT

%°o °o°o°o% o ~°o°o°o°o o-~ ,

o

o

o

~d~L,

IJ1JlllJ v,[.

cF[; LS ,

O

0

o

o

0

IN

Figure 1 Comparison of models for the topology in the membrane of (a) a nicotinic acetylcholine receptor (muscle or neuronal) c¢-subunitand (b) a GABAA receptor l]-subunit. Four membrane spanning domains in each subunit are shown as cylinders (1-4). MA, the predicted amphipathic helix peculiar to the nicotinic receptors, is shown in (a) as part of the long intracellular loop between M3 and M4. That loop is the site of alternative splicing to generate an additional, longer form, known to occur in the GABAA receptor ~2 and l]4 subunits (for references, see Ref. 8) and in the glycine receptor 7 c¢1 subunit. The structure in the extracellular domain is drawn in an arbitrary manner. Potential extracetlular sites for N-glycosytation are indicated by triangles. The encircled P denotes that sites for phosphorylation by protein kinases are present in the intracellular loop in certain receptor GABAA subunits: for kinase A, on all I~-subunits (and on the c¢4 and c¢6 subunits), for kinase C on ?2- (long form), c~4-, c¢5- and c¢6-subunits and for tyrosine kinase on T-subunits. Those charged residues that can be located close to the ends of the membrane-spanning domains are shown as circles with positive charges marked, or as open squares for negative charges. Note the large excess of positive charge which will be at the mouths of the channel when five of the GABAA subunits are assembled to form the receptor. Note also that there will be a small excess of negative charge at the channel mouths in the corresponding structure formed from the nicotinic receptor subunits. Constant cysteine residues (C) are also shown: the vicinal pair in the nicotinic c¢-subunits (only) and the pair 15 residues apart in the 'Cys-Cys loop' in all of the IA receptor subunits (Table III).

where the ? is replaced, presumably by 8 or an extra ix or J~. Further, in at least some cases (see legend to Fig. 1), alternative splicing of the mRNA precursor has been found to generate a second ('long') isoform in each case, which can have an additional regulatory phosphorylation site. This splicing increases the number of subunit isoform types available to at least 18. These are believed, from the heterologous co-expression studies on the recombinant subunits, to be employed in pentameric combinations containing ix, ~ and ?, or ix, 13 and 8, or possibly only ix and ~ types. Hypothetically (based on some sequence features), the p subunits are taken here as additional, specific replacements at an

ix position. Evidence on the actual cooccurrences can be obtained by the localization in situ of the subunit mRNAs and by isoform-specific immunocytochemistry or immuno-coprecipitation. The in situ results show a very complex distribution of many alternative combinations 8,25,26 and even adjacent neurones of the same apparent type can have different combinations 25. In fact, there is recent evidence suggesting that a single location on a neurone can contain more than one ix isoform in the protein 27 (with the [3s not yet tested). The latter type of composition, e.g. pentameric ixlix3~2?2 (of unknown stoichiometry), of which examples appear to be present in a minority of c a s e s 26-29,57, greatly multiplies the theoretical total

371

Signalling at the plasma membrane Table III. Features of the GABAA, glycine, nicotinic acetylcholine and receptor subunits a

TIBS 1 7 - OCTOBER 1992

KA and/or AMPA. The deduced topology of these subunits in the membrane, with four TMs, is very similar to that of Feature GABA and glycine receptors Comparisons with ACh and 5-HT 3 the GABAAreceptor (as in Fig. lb), but receptorsb the amino-terminal domain is more (i) TM2 Contains a common hydroxy-rich This sequence is completely than twice as long. There is also a very sequence Thr-Thr-VaI-Leu-Thrdifferent; an additional acidic low degree of sequence homology of Met-Thr(Ser)- and with a total of groupis near each end of TM2 this series with the neuronal nicotinic eight Ser or Thr in each TM2c ACh receptors, largely in the TM2 domain. However, the other signatures (ii) TM1 Invariant Pro at mid position Invariant Pro at mid position of superfamily ia (Table III) are missing. (iii) TM4 Pro at fourth position, preceded Pro absent Certain forms of these subunits do not by Phe give channel expression on their own, but all do so when combined with an (iv) Charges(per subunit) Always high positive charge A much lower density of positive within eight residues density: up to 13 total charges charges. A small excess of appropriate partner, e.g. KA2 plus negative charges is always of the ends of the TMs, there, and always an excess GIuR633. In general, the observations on the extracellular (up to nine) of positive over present reported suggest that oligomers consides negative charges taining two types of these subunits are needed for the native properties of the Consensus sequenced: (v) ~-Ioopcan form Eight of the 15 positions are identical or very conservatively Cys-X-Hy-X-Hy-X-X-Hy-Probetween Cys139 and range of non-NMDA receptors. Further substituted, in all subunits Hy-Asp-X-[GIn/His]-X-Cys-X-Hy Cys153 heterogeneity is, again, introduced by alternative splicing: P. H. Seeburg and co-workers have shown that GluR1-4 aAbbreviations:TM, transmembrane domain, assuming the structure shown in Fig. 1; Hy, a strongly each exist in two forms (designated flip hydrophobicresidue (here lie, Met, Leu, Val, Tyr, Phe or Trp); X, any amino acid. The numbering is for the GABAreceptor czl subunit. and flop) which differ by a 38-residue bNicotinic acetylcholine receptor subunits4 from electric organs, muscles and neurones and the insertion in the large putative intraneuronal 5-HT 3 receptor 19, that is, the cation channels. cellular loop 34. These two forms differ in CTheglycine receptor [3-subunitis distinct from all the others: in its TM2 there are only five Ser or their channel properties, when glutaThr residues, and the first, fifth and seventh Thr residues of the common sequence are changed mate is the agonist 34. there to hydrophobicresidues. din all of the known subunits of the GABAA, glycine, nicotinic ACh and 5-HT3 receptors. An important molecular distinction35,36 between the channels relates to their permeability to Ca 2÷. This is very low in of receptors. Considering restrictions non-NMDA receptors. In the latter class most (but not all) native non-NMDA on some co-occurrences that are ap- the agonists kainate and AMPA (c¢- receptors tested in situ, but in recomparent from the observed distributions, amino-3-hydroxy-5-methylisoxazole-4- binant homomers and heteromers of this leads to the order of 1000 possible propionic acid) can distinguish some of GIuR1, 3 or 4 it is high (Table IV). When a GluR2 subunit is substituted it becombinations (or -4000 if two 13 iso- these in situ 3°. Non-NMDA receptors. Oocyte expression comes low again; this control is exerted forms can also be present). Possibly 50-100 of these actually occur, from the cloning by S. Heinemann and co-workers by an invariant Arg in the TM2 limited knowledge so far obtained by yielded the first structure of a subunit sequence of GluR2 (replaced by gluta(GIuR1, 101 kDa) w i t h non-NMDA recep- mine in the others) (Table IV). The the aforementioned methods. tor properties 31. Starting from this, a genes in each case contain a Gin codon family of homologous subunits has at this site, which is 'edited' at the The ionotropic glutamate receptors The transmitter-gated (i.e. ionotropic) been obtained: GIuR1-7 and KA1, KA2 mRNA (or conceivably DNA) leveP7, as (Refs 31-33 and references cited there- another level of regulation of these glutamate receptors are clearly divided functionally into those activated by in). These differ functionally in their channels. This evidence supports the conNMDA (N-methyl-D-aspartate) and the channel responses to, and affinities for, cept of TM2 as a channel-lining domain. 5-HT 3

Table IV. Calcium ion permeabilities of cationic channels of receptors a ACh Muscle

Glu (non-NMDA)

Neuronal GluR1-4b

Glu (NMDA)

GluR2(Q)c

GluR2(R)c

NRI+ NR2Ad

Pca/Pcs

0.2

1.3

1.2

1.2

0.05

2.4

References

55

55

35

35

35

35

aThe ratio of the permeability of Ca2+ to that of Cs+ (Pca/Pcs) is an experimental measure of the Ca2+ permeability of the channel, and is shown for the nicotinic and glutamate receptor types (native or recombinant) listed. bwith any of these as homomeric receptors. cWith the Arg in the TM2 regioneither present (R) or replacedby Gin (Q)35,36.The homomericvalue in each case is not changed when either is made heteromeric with GluR4 (with its natural Gin there). dAs a heteromer. The value here is close to that for native NMDAreceptors in the same conditions.

372

NMDA receptors have in recent years attracted the greatest attention in functional studies of the transmitter-gated

ion channels. They have many properties that are unique in that class, including sensitivity to voltage as well as to transmitter, the requirement for a coagonist (glycine), slow kinetics, high permeability to Ca2+ as well as Na + and K*, blockade by physiological levels of Mg2÷ and an exceptional range of allosteric modulatory sites. A landmark in this field has been the determination, again by oocyte-expression cloning, of the structure of a subunit of a rat NMDA receptor by Nakanishi and co-workers 3s. This poly-

TIBS 17 - OCTOBER 1992

Signalling at the plasma membrane

peptide (NR1) of 105 kDa can be expressed as a homomer which, surprisingly, is sufficient to produce all of the aforementioned features characteristic of the NMDA receptor. This subunit is only -22% identical in sequence to GluR1-6 or KA1 and KA2. It has, however, four predicted TMs which correspond in position and sequence similarity to those commonly recognized (although two viewpoints are noted in Ref. 30) in GluR1-6. Starting from the NR1 DNA sequence, clones encoding a number of related NMDA receptor subunits have recently been obtained in several laboratories. Three of these have so far been fully described: NR2A, NR2B and NR2C by Mishina and co-workers 39,4° (who term them ~1-3), and by Seeburg and coworkers 41 [although their rat form of NR2C lacks a 277-amino acid stretch at the carboxy] terminus that is present in the mouse (e3) form4°]. None of these has produced, in expression studies with agonists, a homomeric channel, nor in their combinations, but each forms a heteromeric receptor channel with NR1. Interesting features of this system include: (1) the subunit lengths are no longer similar, with the NR2 sizes ranging from 133 to 163 kDa, all of the excess over the 103 kDa of NR1 being in a completely variable carboxyterminal extension; (2) there is only -18% sequence identity with NR1, but 40-52% between the NR2s; (3) the heteromers differ considerably in their Mg2÷ sensitivity and in some pharmacological properties. The high Ca2+ permeability characteristic of NMDA receptors in situ is reproduced in them (Table IV). Types of glutamate receptors. The subunits can be classed, as Mishina and coworkers have pointed out 4°, into groups of related sequences: ((z) GluR1-4; (13) GluR5-7; (y) KA1 and KA2; (4) NR1; (e) NR2A-C. To these we should add the two invertebrate types so far known, molluscan 42 and Drosophila43non-NMDA receptor subunits. These show only 3746% or 25-28% identity, respectively, with GluR1-5, and only 26% identity to each other, but otherwise they exhibit the general features of GluR1-7, so that they appear to represent two further non-NMDA types. The classification of the o¢, [~ and y types according, also, to their selectivity for AMPA and kainate 39 no longer holds, since the y type can combine with a [3 type to change from kainate to AMPA responsiveness 33.

In considering the channel structure, it is important to note that subunits which are very remote in sequence comparisons and of very different sizes combine to form the NMDA receptor. Only some putative transmembrane regions are homologous between the NR1 and NR2 sequences, but this appears to be sufficient to allow substitution of one type by another to form an active heteromer. This feature would allow also, in principle, the substitution in (say), an (~ type receptor of an NR subunit, to give a unitary NMDA/nonNMDA receptor type. Evidence to show that this can indeed occur has been obtained in the case of Xenopus receptors 44, where the o¢type of subunit has a molecular mass of only48 kDa. Overall, while the glutamate receptors show an unprecedented degree of sequence diversity, the local (TM) homologies and the universal pattern of hydrophobic domains support the concept that they are all - NMDA and non-NMDA types - in one superfamily, designated Ib on account of its distant relationship to the nicotinic receptors. Not only do all the la and Ib channels show evidence for a similar topology, but they also all contain a long loop between TM3 and TM4 which offers numerous potential sites for intracellular regulation. However, the addition of a large carboxy-terminal domain in the NR2 subunits signifies that the deductions on the transmembrane topology in this series are still only presumptive. The great diversity found in the limited set of glutamate receptor subunits so far known also indicates that generalization on their channel-forming mechanism is premature. It is clear that a multiplicity of glutamate receptors can exist, as many as those for GABAA, and for each far more than for any other known receptor type. GABA and glutamate have in common their requirement in fast signalling and their ubiquitous use throughout the brain. For both, it must be more efficient to use a large number of channel types, obtained combinatorially from 20 or more isoforms, so that a single, simple transmitter molecule can be applied in a great variety of distinctive neural pathways and developmental mechanisms.

Acknowledgements Parts of Tables I-lIl and Fig. 1 are taken, with permission, from Chapters 4 and 6, by E. A. Barnard, in Receptors Subunits and Complexes (A. Burgen and

E. A. Barnard, eds), Cambridge University Press, 1992.

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ALL RECEPTOR TYROSINE KINASES

(RTKs) that have been studied phosphorylate themselves on tyrosine (autophosphorylation) in response to ligand binding. Several experiments over the past three years have highlighted an important function of autophosphorylation: individual phosphotyrosine residues of receptors appear to serve as highly selective binding sites that are specific for cytoplasmic signaling molecules. These signaling molecules mediate the pleiotrophic responses of cells to growth factors. Most of the studies have used the platelet-derived growth factor 13-receptor (PDGF[3r) and fibroblast growth factor receptor (FGFr). When activated by a ligand, the intracellular region of the PDGF[3r binds several signaling molecules, including phosphatidylinositol 3-kinase (PtdIns3-kinase), GTPase-activating factor (GAP), phospholipase C~/ (PLCT) and c-Src (for review see Ref. 1). Receptor mutagenesis studies demonstrated that PtdIns3-kinase binds to two distinct sites on the PDGF[3r (Tyr708 and Tyr719 of the murine receptor) 2-4 while GAP binds to a third site (Tyr739) (Fig. 1)4,5. PLC~' binds to Tyr766 of a human FGFr6,7 and to two phosphotyrosines in the carboxy-terminal portion of the PDGFJ3r (Fig. 1; L. R6nstrand, submitted). Determining the structural basis for the specificity of the interaction between RTKs and signaling molecules has established what is likely to be a general principle of how tyrosine-phosphorylated proteins bind regulatory molecules. Phosphotyrosine-containing peptides as short as five amino acids, M. J. Pazin and L. T. Williams are at the Howard Hughes Medical Institute, Program of Excellence in Molecular Biology,and Cardiovascular Research Institute, University of California, San Francisco, CA 94143-0724, USA.

374

Growth factor receptors that are tyrosine kinases (RTKs) regulate growth and differentiation of cells in many organisms, including flies, worms, frogs, mice and humans. There has been recent progress in understanding the mechanism by which these receptors transduce signals. Worm and insect studies on RTKs have relied primarily on genetics, while the mammalian studies have employed a combination of molecular genetics and biochemistry. While many RTKs seem to have unique features, there are also many general signal transduction principles that emerge from these studies. In this review, we will focus on common signaling molecules, using RTKs from both vertebrates and invertebrates as examples.

representing RTK sequences at the signaling molecule-binding sites, can selectively block the interaction of signaling molecules with RTKs5. The binding of peptides to signaling molecules is highly specific and occurs at low concentrations of peptide (1-10 Bm), but only when the peptide is phosphorylated on its key Tyr. For example, the peptide YPVPML, where YP represents phosphotyrosine, is a PDGF[~r PtdIns3-kinasebinding site that blocks PtdIns3-kinase binding to PDGF[~r but does not block two other signaling molecules, GAP and PLCy5. YPMAPYDNY, which represents the GAP-binding site on the PDGF[3r, blocks GAP binding and has no effect on the binding of PtdIns3-kinase or PLCy, and SNQEY2"LDLS, representing the FGFr PLCy-binding site, blocks PLC~, binding to FGFr and to PDGFJ3r6,7 (Table I). In separate experiments, the specificity of the peptide interaction was confirmed by mutating the Tyr or Met residues in the PDGF[3r PtdIns3-kinasebinding site, expressing the mutant receptor in fibroblasts and epithelial

cells, and demonstrating that these mutations cause a loss of PDGF-stimulated activation of PtdIns3-kinase 4,5, Motifs homologous to the PDGFJ3r Ptdlns3-kinase-binding site are found in other RTKs, including the macrophage colony stimulating factor receptor (MCSFr). Mutations of the Tyr in this motif cause a loss of PtdIns3-kinase binding to the M-CSFr8. These studies indicate that short sequences flanking receptor phosphotyrosines are major determinants of the remarkable binding specificity of signaling molecules to RTKs.

SH2 domainsand bindingto RTKs The region of the signaling molecules that binds RTKs has been characterized. Hanafusa and co-workers demonstrated that SH2 domains (domains homologous to a non-catalytic region of the c-src proto-oncogene) will bind directly to Tyr-phosphorylated proteins in transformed cells 9. Most of the signaling molecules known to bind RTKs contain SH2 domains. Although at high concentration (achievable in vitro) or in

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