The molecular basis of odor recognition

TIBS 1 2 - February 1987

63

The molecular basis of odor recognition Doron Lancet and Llmberto Pace Following decn,,l~ of sauty and spectdamm, the molecula, mechamsms of olfactton are begin. tung to be tmdersfood Odorant receptors appear to actwate a cyclic nucleolute enzyme cascade, including a G TP.bmdmg protein, analogous wuh the processes of hormone, neurommmutter and vmedreception A typical olfactory epithelium contmns 107 structurally similar, bipolar sensory neurons with long clha emanating from their dendmes (see Anholt's accompanying amcle, Fig 1) Olfactory ctha are the site of sensory transductton, akin to visual rod and cone outer segments Different sensory cells gwe the same response to different odorants depolarization and the finng of action potentials 1 2. Thus, it was suggested that different olfactory neurons express distinct olfactory receptor (OR) molecules, which activate a common transducUon chain (Fig 1)

Olfactory transdectien: cyclic AMP cascade One of the rewarding recent developments tn olfactory biochemistry has been the eluadatmn of an olfactory transductlon mechanism In analogy with neurotransmitter and hormone slgnahng in neurons and other cells3 4, and with hght activation of photoreceptor cells 5 (Fig 2), accumulating data suggest that cyclic AMP (cAMP) serves as second messenger in odorant activation of the chemosensory neurons Electrophyslologlcal studies show that cAMP (or its membrane penetrable analogs), phosphodtesterase inhtbltors, and guanine nucleotldes can ehclt odorant-hke responses or modulate odorant activation t 2,6 7 Biochemical studies, carried out in cdmry-ennched membrane preparations (Refs 8 and 9 and references cited thereto) analogous to isolated retinal rod outer segments, confirm and extend these results The isolated chemosensory organelles of frog9-n and rat tl-i3 contain a very high specific activity of adenylate cyclase, and the clhary enzyme is actwated 1 5-2 5-fold by odorants at physiological concentrationst°-~ This odorant response is tissue- and hgand-specafic and GTP-dependent, as D Lancet and U Pace are at the Department o f Membrane Research, The Wet27nann Insumte o f Scteuce, Rehovot, Israel

expected for a second messengergenerating enzyme coupled to a specific receptor wa a GTP-bmd, ng protein (G protein)3. 4 Odorant rmxtures are more efficient than mdwldual r,oorants in activating adenylate cycla~¢ ~0, consistent with the notmn of several O R molecule classes converging or~ a common transductlon mechanism Additional corroboration of receptor heterogeneity comes from the low value of Hill's coefficient (~0 4) displayed by the odorant activation doseresponse curves t° Adenylate cyclase measurements m isolated olfactory ctha prowde the first cell-free assay for olfactory actwatton 214 Sklar e~ a/If were able to conduct a rapid screening and comparison of

sensory cell types

S1,S2

the activation properhes of many dozens of odorants usm- ,he assay (see 'Other transduction mechanisms') This assay also showed that gp95, a major transmembrane glycoprotem of olfactory cihas 15 16, might be involved in odorant reception (see 'Olfactory receptor isolalion') An obwous advantage of the adenylate cyclase assay over ligand binding techniques is that only functionally Important interactions are registered Non-specific binding, which presumably does not lead to transductlon events, remains undetected Ogactory G protein The GTP dependence of odorant activation suggests that guanine nucleotldebinding protein (G protein) is involved in the couphng between OR and adenylate cyclase Olfactory G protein appears to be similar to G~, the stlmulatory G prorein of some hormone and neurotransmitter receptors ~, since odorants enhance rather than dmunish cAMP production Also, cholera toxincatalysed [32p]ADP nhosylatmn of olfactory cdsa of frog 1017 and rat I-' labels a polypeptlde substrate of 42-45 kDa resembling the o-subumt of Gs, and leads to actwatmn of adenylate cyclase in the clhary membranes r- The same G,, polypeptlde can be seen through interac-

S1,S3,S4

S5

•-.

S2,S N

o,,oc,o

receptor proteins second messenger generation

-

GTP GDP-,"

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membrahe transduction Fig i A schemntw view o f olfactory receptwn ,4t least a fe,~ dozen (M) olf~ctor~ receptor molecule IORJ types may exmst, present on the dendrites o f a similar number IN) o f ~ensor~ neuron t~pes (see Refi 2 26 and 37) A sensor, neuron ( e g SI~ may have more thml one t~pe o f receptor molecule (m this case O R I and O R ~ A given OR molecule ( e g OR,_) ma, be present on more tha, r one semorv neuronal tvpe (m this case S t, St and S.O Altemauvely, clonal exclnslon may pertain, where a one-to-one relauoashrp adl hold fe g sensory neuron 5~ and O R molecule O R # Many O R molecule types ma~ com erge (m dtfferem cells) on a common trarL,ducuon machinery, the best candrdate being G~ and adenvlate c; close (see re:Ill c 4 M P for other messengers produced b) parallel transduruon en."vmes ) tt ould then actt; ate the sensorz trot channels to produce neuronal membrane depolanzauon ~ ) 19~7 El,,c,,zer Science Pubhsht.r~ B ~

AmslO'dam

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64

=ion with G prote[]-specttic antisera t4,=7 In flog, olfactory Gm, hke adenylate cyclase ts enriched [] olfactory ctha compared to membranes from hver or from decthated eptthehum t° 12 t7 Other G protein types, the intubttory (G~) and the brmn-speafic (Go) are present I° 12, but are not parucularly enriched m olfactory ctha These prote[]s could be related to modulation of the odorant response, possible by endogenously secreted hgands is Gs has been shown to eyast [] at least two molecular forms, with a-subumts of 42-45 kDa and 48-52 kDa ~. produced by alternative mRNA sphcmg 19 Olfactory csha con=am mainly an ADP-nbosylated a-subantt sandar, but possibly not identical to, the lower molecular weight form 12 Olfactory G s appears to differ from G, m brmn or hver [] having higher guan[]e nucleottde affimty 1° 12 and different relative potency of actlvauon by stereolsomenc gua[]ne nucleonde analogs 14 Such differences could be due either to gene-related ammo acid sequence vanauons or to post-translational modtficatmn Polynucleoudes coding for G protems [] a rat olfactory epithelial eDNA hbrary have been sequenced, and clones corresponding to G s, Gt and G o, as well as at least two other G proteins, identified 2U At present tt ~s not clear wluch of these sequences corresponds to the functmnal component of the sensory neurons Interest[]g support for the proposed role of G s [] olfactmn comes from studies of the human geneuc deficiency disease, pseudohypoparathyroichsm (PI-IP) PHP type Ia patients, who are reststam to the action of several cAMP-mediated hormones, and have only about 50% of the normal G~ level, are tound to have an imp•red olfactory capacity 21 It is suggested that the sensory impairment =s due to a defect [] the peripheral receptmn mecha[]sm lfso, these data may be taken to imply that olfactory G Sts either =denucal with hormonal G s, or that both are under common genetic control Other transduction mechanisms Is adenylate cyclase activation the only mecha[]sm of olfactory transduc=ion9 Many odorants (notably those [] the fruity, floral, mmty and herbaceous classes) activate adenylate cyclase, but other physiologically active odorants (e g putrid ahphatm .,ctds, ammes and orgamc solvents) do not affect the enzyme in ,solated clha tt Thus, certa[] OR molecules may generate an lntracellular signal through different transductmn mechanisms Guanylate cyclase and cyclic nucleotide phosphodlesterase

T I B S 12 - February 1987

in rat olfactory eptthehal membranes were found to be odorant []sensitive t3 Another possible mechamsm ss modulation of phosphattdyl •osttol metabolism 22, supported by ewdence whereby L-alamne, an anuno acid odorant, activates phosphatidyl mosRol-4,5-b=sphosphate phosphodmsterase (phosphohpase C) in isolated fish olfactory clha23 There are other possible transductlon mecha[]sms (1) The direct modulation by some odorants of ton channels m the sensory neuronal membranes 24, stmdar to the mechamsm of action, of the •cot=[]c acetylchohne receptor (see Anholt's article for a further discussion) Such a mechamsm lacks the amphficauon provided by second messenger enzyme cascades, but may be s=g[]ficandy faster* (2) Receptor-mdependent odorant modulation of the transduction machinery, e g direct activation of olfactory G protem and/or adenylate cyclase proteins by tluol reagents Is The latter could account for the potent and rather umform odor of most suL~ydrylcontmnmg odorants Olfactory receptor diversity An mterest[]g proble[] =s how so many odorants with widely diverse []olecular structures are agomsts, t e are capable of []ducmg the correct conformat,onal transmons necessary for receptor activatmn in other receptor systems, only a few []embers of any given group of structurally related hgands can serve as agomsts In contrast, practmally any chemmal modification of a gwen odorant will yield another odorant, though often with a different perceived quality Two possible explanations are. (1) OR molecules have partmulafly broad selectmty ranges, (2) there ts a rather large number of different OR protein types Because of the stnlong resemblance between olfactory transduction mechamsms and those of other G protem-acttvatmg receptors, it ~s less probable that OR molecules are unique [] thew allostenc properties It appears more hkely that OR molecules constitute a repertoire of many different receptor types, each with the usual narrow ago•st range It has been postulated that OR protems constitute products of a multigene family, similar to that of =mmunoglobullns (see Ref 2 for a review) Members of this famdy, would be expressed [] different sensory cells, either in a 'one cell one receptor' type arrangement or *The speed of the olfactory response (100-1(300 ms) is consistent with both a d.¢ct mechanism and a second messenger cascade2

otherwise, accountmg for the dwerstty of odorant responses of []dmdual sensory cells Like tmmunoglobuhns and T-cell receptors 25, OR molecules could have variable regions contaimng the odorant bmdmg rues and share a constant regmn responmble for transductton (Fig 1) What is the possible raze of olfactory r e c e p t o r r e p e r t o i r e 9 Data on human specific anosnua~ (genetic defects [] the abthty to perceive certmn odorants) mdlcate the existence of at least a few dozen receptor types26 The variations of electrophysmlogmally momtored s[]gle-cell responses in the olfactory eplthehum are consment with this nummum number=,2 Based on hgand affimty cons,deratmns tt has been argued 2as that the upper Imut for the olfactory repertoire may be 102-104 , considerably smaller than the tmmunoglobulm riper=one (107-109, Ref 25) lntngumgly, color vmon operates with only three photoreceptor type s27, possibly because absorption spectra of orgamc chromophores can easily cover one-third of the entire v~sible wavelength range, wlule a typical protein receptor would usually b[]d only a much smaller fraction of all possthle hgandst Olfactory receptor isolation The neuronal membrane receptors that transduce odorant signals have not yet been unequivocally identified The difficulties [] acluevmg tins goal by hgand-blnd[]g techmques may be attnbuted to the possible dwerstty of OR blnd[]g sRes, as well as to the expected high levels of non-specific blnchng due to odorant hpophthclty and presumed weak affi[]ty2. A pyrazme-b[]d[]g protern isolated from olfactory eplthehal tissue was found to be water-soluble and present m relabvely Ingh concentrations in secretory glands and mucus of nasal epltheha (Ref 29 and references cited therein) It probably acts as an odorant career or scavenger facdttat[]g access to or removal from the receptors An olfactory eptthehal camphor b=ndmg activity3° remams to be identified as a defined polypeptide species An alternative approach to the identification of OR proteins ts to screen the sensory ctha membranes for polypeptide candidates with receptor properties other than odorant blnchng Useful cntena can be those features which are common to other G protem-acttvat=ng receptors trans[]embrane disposition, =Once a photon ,s absorbed, photo~somenzed reunal may mduce a similar conformaUonal change to that caused by odorant bmdlng Notably. many potent odorants are ,soprenoid compounds, similar to retmal ~

TIBS 12 - February 1987 glycosylatron ,

tissue specticq and mteracnon wth G, and adenylate cyclase A promismg receptor can&date IS glycoprotem gp95, the only specific polypeptlde of frog olfactory cdla that fulfills many of these cTytena2,s1516 This glycoprotein IS a major diary component, whose bdayer concentration agrees Hnth c@lunts of freeze fracture intramembranous partlcles suggested to correspond to OR moleculessl Electrophyslolog& and cell-free adenylate cyclase studies employmg specific lectms and antIbodIes provide support for Its mteractum wth the olfactory transduction machmeryl4 Homologs of gp95 appear to be present m other vertebrate speaesl6. and its future molecular genetic charactenzatlon may provide Important clues on olfactory receptio&Is Specifically, it wdl be important to determme whether gp95 (or any other receptor can&date) displays heterogeneity at the protein and gene levels

65 Involved, or about the mechamsm of theu modulation by CAMP 0ao1 shmulatlon leads to depolanzatron and increased membrane conductance In olfactory neurons’, suggestmgthe opening of catlon channels The best candldate for the transduction current tamer IS sodium. but potassium has also been Implicated (see accompanymg review by Anholt) In addition to actmg through phosphorylation by CAMP-dependent protem kmase, CAMP could act through duect allostenc modulation of Ion channels, in analogy wth the function of the cGMP-gated sodium channels of retmal rod outer segment (reviewed m Ref 8; Fig 2) Evidence for the latter mechamsm has recently been obtained through smgle ion channel recordmgsU

Photon

Olfactory adaptation Electrophynolo~cal signals recorded from the penpheral sensory neurons of olfactory eplthebum exhlblt partial adaptation wthm 1 s from the onset of stimulus Tlus processappears to be dlstact from the well-known disappearance of odor sensation upon longer (- 1 mm) exposure, which IS thought to be mediated by central nervoussystem processing Fast olfactory adaptation may be akm to desensltlzatlon processes In other G protem-linked receptors In other systems,the photoreceptor protein rhodopsm and P-adrenerguz receptor, molecular detads of such adaptation processeshave been elucrdated(renewed III Refs 5 and 35) Smcc OR molecules appear to share functional propertles with both P-AR and rhodopsm (Ref 2.

B-Agonist

Odoront

I FramcAMPtoionchuds The major mtraceuular target of the second messenger CAMP IS CAMPdependent protein kmase. ‘IIus enzyme IS present m olfactory &a and m declhated epithelral membranes32 It is achvated by nucromolar concentrabons of CAMP, and by GTPyS, which presumably acts through endogenous CAMP generation (cGMP IS a much lesspotent activator ) Several clhary polypephde substrates, notably two with molecular masses of 24 and 26 kDa (pp24 and pp26), have been ldentlfied through their CAMP-dependent phosphorylatlonJ2 These results stimulate further studies of the mvolvement of CAMPdependent protein phosphorylatlon m olfactory transduchon m parallel to its role m other neuromodulatlon processes33 The other can&date transducmg enzyme, phosphohpase C, hberates two second messengers, mosltol tnsphosphate and Bacylglycerol, the latter actlvatmg protein hnase C (Ref 22) Such kmase actlvlty was Investigated In olfactory membranes, with confhctmg results Anholt et al I7 Idenhfied protem kmase C a frog olfactory cdla by lmmunoblot analysis and by phorbol ester bmdmg However, neither they, nor Heldman and Lancet”, could demonstrate protein kmase C atinty by phosphorylatlon assays m the presence of calcium and phosphatldylsenne Elect,ophysdo@cal and blochenucal data support the notion that changes of mtracellular CAMP modulate membrane Ion conductance Yet, httle IS known about the nature of the Ion channel(s)

Rh 3

9ARK

w Gs

GMP cGMP4

ATP

?g

cAMPt

AC

ATP c+MPt

No /Ca t

Fig 2 A hypothettcal view o/rheposstble homology between o@cforv r~eplor (OR) molecules and orher cychc nucleonde-coupled receptors. bdrenergrc receptar @AR) and the phororemptor protem rhodopsrn (Rh) Odomnt moleculesserve an analogous role to ~gonrsts or to photons, acavanng a ret umdenn)ied OR protein OR protems may belong to the group of membrane receptors wth seven transmembmne domarnr that mcludes BAR and rhodopsm IS Ltke pA R, OR protemacnvatea G,-adenylate qclase svstem rncreasrngtheproducnon of CAMP/mm ATP Analogoudy. photolysed rhodopsvl acts aresa transducmphosphodrestemsesystemto mcrease thebreakdown of cGMP Two dtstmctmechanismsmediate the modulanon of wn channel conductance bv cychc nucleotrdes In some neurons, CAMPmav causethe opening or ton channels bv acnvatrng CAMP-dependent prorem kuaase whtch catalysesprottw phosphonlonons’ In rod outer segmentscGMP activates a canon conductance bv dwectly mtemcnng with an mn channel’ One or both of thesemechanwm may underhe theodomnt-related acttvatton of a mnon conduaancem dfactov neurons OR protetns could undergo phosphorylanon bv an oljacton receptor-spenfic kmaw” (labelled ORK). hypothesrzedto be homologousto PAR kmase @ARK) and to rhodopsm krnare (RhK)

66

Fig 2), It would be mterestlng to look for phosphorylatlon of olfactory cilia polypepttdes by receptor-specific klnases that act only on activated receptors 35 In parallel, c A M P - d e p e n d e n t p r o t e m lanase and protein k m a s e C could phosphorylate transductlon comp o n e n t s a n d play a role in c h e m o s e n s o r y adaptation (see Ref 35) Conclusion F u t u r e research of olfactory reception will probably culminate in the Identification, ~solation and study of O R molecules A most r e w a r d m g result of such a c c o m p h s h m e n t could be a complete m a p p i n g of O R genes m the h u m a n g e n o m e , helping to elucidate the molecular genetic basis o f specific olfactory deficiencies a n d p o p u l a t i o n polymorphlsms, slmdar to the recent achievements m the field o f color p h o t o reception 27 Because there m a y b e 10100 times more genes Involved, the degree of indlwdual v a n a t m n In O R genes m a y be extreme, possibly comparable to that found in lustocompaUblhty genes which have been shown to be functionally related to odor reception 36 Studies of O R genes can greatly benefit from research o n D r o s o p h i l a m e l a n o g a s ter speclfic a n o s m l a m u t a n t s 37 E x t e n d i n g the biochemical and geneuc studies of O R molecules to insects m a y provide a crucial source of knowledge for the study of the mechanisms invoh,ed In insect p h e r o m o n e detection 3s T h e example set by studies of olfactory r e c e p t m n may pave the way to a molecular eluc~datlon of other chemoreceptaon systems notably taste and vomeronasal reception, both d e a h n g vath sttmuh delivered m a q u o , the latter possibly constituting a 'pept~derglc' olfactory-hke system 39 Unraveling O R genes may lead to a b e t t e r understanding of gene expression m developing or regenerating olfactory sensory neurons This should help resolve o p e n questions such as how olfactory neurons form the correct s y n a p t ¢ contacts depending o n t h e i r O R specificity 2 Thus olfactory n e u r o n s may b e c o m e a useful model for studies of n e u r o n a l connectivity and function as well as for molecular transductlon mechamsms

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T I B S 12 - F e b r u a r y 1987

671-677 7 Pe~aud. K C . Heck. G L and DeSimone J A Nature(repress)

8 Chen Z and Lancet. D (1984) Proc Natl Acad So UfA 81. 1859.-1863 9 Chen Z . Pace. U . Heldman. J Shaptra. A and Lancet. D (1986) J Neurosct 6. 21462154 10 Pace. U . Hansh. E . Salomon. Y and Lancet. D (1985) Nature 316.255-258 11 Sldar. P B.Anholt R R H andSnyder. S H (1986)J Biol Chem 261. 15538-15543 12 Pace. U and Lancet. D (1986) Proc Nad Acad Set USA 83 4947-4951 13 Shirley. S G . Robinson. C J . Dickenson. K . Aulla. R and Dodd. G H (1986) Biochem J 240. 605-607 14 Lancet. D. Chen. Z C~obutano.A . Eckstem. F Khen M. Heldman J Ophw.D. Shafir. i and Pace. U Ann NYAcad So (repress) 15 Chen Z . Oph,r D and Lancet. D (1986) Brain Res 368.329-338 16 Chen Z . Pace U . Ronen. D and Lancet. D (1986)J Btol Chem 261.12~1305 17 Anholt R R H . Mumbv S M Stoffers. D A Gnard P R Kuo J F Gdman. A G and Snyder S H (1987)Btochermstrv (m press) 18 Lancet D m Molecular Biology of the Olfactory System (Margohs. F L and Gelchell. T V. eds) Plenum Press (m press) 19 Roblshaw. J D Grazmno. M P. Sin:gel. M D andGdman. A G (1986)J Btol Chem 261 9587-959O 20 Jones. D and Reed. R Chem Senses (m press) 21 Wemstock. R S. Wnghl. H N. Spiegel. A M .Levme. M A andMoscs. A M (1986) ~'amre 322.635-636 22 Bemdge M J andlr~,ne R F (1984)Nature 312 315-321 2"; Huque T and Bruch. R C (1986) Bfophys

Biochem Res Commun 137,36--42 24 Vodyanoy. V and Murphy. R B (1983) Science 220. 717-719 25 Hunkapdler, T and Hooa, L (1986) Nature 323, 15--16 26 Amoore J E (1971) m Handbook of Sensory Physiology (Vol 4. part l) (Beidler. L M , ed ). pp 245-256, Spnnger-Verlag 27 Nathans, J , Thomas, D and Hogness, D S (1986) Science 232, 193--202 28 Boelens, H (1982) tn Fragrance Chemtstry (Thelmer, E T , ed ), pp 123-163, Acadenac Press 29 Pevsner, J , SHar, P B and Snyder, S H (1986) Proc Nail Acud Sct USA 83, 49414946

30 Fesenko, E E , Novoselov, V I and Nov]kov. J V (1985)Btochlm Btophys Acta839.268--

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36 Boyse, E A , Beauchamp. G K , Yamazakl. K, Bard, J and Thomas, L (1982) Oncodev Biol Med 4.10l-116 37 Rodngues, V and StdchkJ. O (1978) Proc Indian Acad Scl . Sect B 87, 147-160 38 Katsshng, K E (1986)Annu Rev Neuroscl 9. 121-145 39 ~rshenbaum, D M , Shulman, N and Halpern, M (1986) Proc NatlAcad Scz USA 83, 1213--1216

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