- Email: [email protected]
The second messenger cascade in olfactory receptor neurons
The second messenger cascade in olfactory
receptor
neurons Heather Howard
Hughes
Medical
A. Bakalyar
Institute,
and Randall
The Johns Hopkins
School
R. Reed
of Medicine,
Baltimore,
Maryland,
USA
The molecular cloning of components involved in the CAMP second messenger cascade has allowed their biochemical characterization and revealed properties that are important for their role in sensory transduction. Recent evidence suggests inositol 1,4,5-trisphosphate functions as an additional second messenger in olfactory signalling. The interaction of these two pathways may contribute to the sensitivity of the olfactory system. Current
Opinion
in Neurobiology
tribute to the overall sensitivity and specificity associated with odor detection.
Introduction Olfaction is the process by which information
regarding the external chemical environment is transduced into an electro-chemical signalling cascade which leads to an action potential encoding for the identity and intensity of the original stimulus. In mammals, odorants (compounds which can be perceived) are drawn through the nasal lumen, solubilized and brought into direct contact with the olfactory receptor neurons. Long, non-motile cilia on the apical membrane of these bipolar neurons project into the mucus layer which bathes the surfa* of the olfactoIy epithelium. These ciliary structures, exposed to the external environment, are the site of the primary olfactory events: odorant recognition, transduction of the external stimulus into an internal chemical signal and subsequent membrane depolarization [ 11. Membrane depolarization initiates an action potential which propagates the signal to second order neurons in a specialized processing region of the brain, the olfactory bulb. Psychophysical studies have demonstrated that the human olfactory system is able to discriminate between more than 10 000 odorants with high sensitivity, some airborne molecules can be detected at concentrations as low as a few parts per trillion
[21. The sensitivity attributed to this system is at least in part achieved by a signal-amplifying second messenger cascade. A GTP-dependent increase in adenylyl cyclase activity in response to ‘fruity’ odorants like citralva is obsend in olfactory cilia. This increased enzyme activity results in a transient rise in CAMP concentration that precedes odorant-induced membrane depolarization [ 3~=]. Several components of classical GTP-binding protein (G protein) signalling cascades (a G, subunit of the G protein heterotrimer, an adenyiyl cyclase, and an ion channel gated by a cyclic nucleotide) appear to have evolved specialized counterparts in the olfactory system [4,5**,ti*]. Functional interaction of these components may con-
C protein---GTP-binding
204
1991, 1:204-208
@ Current
Recently, Breer’s laboratory [7**] has demonstrated that the odorant pyrazine produces an intracellular rise in a different second messenger, inositol 1,4,5-&phosphate (IP$, in rat sensory cilia. This implies that a second messenger pathway other than CAMP, responsive to different odorant stimuli, may be involved in mammalian olfaction. The mechanism by which these separate second messenger olfactory cascades may be integrated in the complete olfactory process is as yet unknown, although a role for Ca2 + -activated calmodulin has been hypothesized [ W]
Odorant interaction generation
to second
messenger
In mammals, airborne odorants must diffuse into the mucus layer bathing the nasal epithelium prior to contact with recognition sites on the olfactory neuronal cilia. Odorant-binding proteins may play a role in concentrating some odorants and in presenting odorant molecules to the recognition sites. The observed negative cooperative binding of odorant ligand to odomnt-binding protein may facilitate the uptake of odorant from ambient air in the nasal lumen and its subsequent release in the mucus layer (9*]. Loose patch-clamp recording studies on in situ olfactory receptor neurons indicate that ionic conductances appear in response to picomolar concentrations of applied odorant [lo*]. Once the odorant has reached the olfactory receptor neuron surface, interaction with the recognition site, presumably a protein receptor molecule, takes place. The mechanism conferring specificity to this interaction has yet to be elucidated; a large number of highly specific receptors may be involved, or a smaller population of unique recep-
Abbreviations protein; IP3-inositol Biology
prior
1,4,5-trisphosphate.
Ltd ISSN 09594388
The second
messenger
tom with broadly overlapping specificities may act combinatorially to discriminate chemically distinct odorants [ 1,11*,12*]. The recently described multigene family encoding olfactory-specific, seven transmembrane domain proteins may encode the repertoire of putative odorant receptors [ 13**]. These molecules may prove useful for investigating the origin of specilicity in odorant detection.
The mechanisms generation
of second
messenger
Kinetics
Firestein et al. [ 14.01 have employed a whole-cell patchclamp technique to delineate the time course of the salamander olfactory neuron response to odorant. The application of odorant pulses, directed at the cilia, elicited a current after the concentration-independent latency penod of approximately 500 ms. The magnitude of the current response was a function of both stimulus duration and odorant concentration over a dynamic concentration range of approximately one order of magnitude. Short odorant pulses (less than 1OOm.s) generated a current that peaked after odorant had dilfused to negligible lev els. These observations are consistent with the presence of an integrating step, for instance, the generation of second messenger, in ciliary odorant-induced membrane depolarization [ 14**]. Modulation of cAMP concentrations in rat olfactory cilia was shown to follow the kinetics expected for such a second messenger in olfactory signal transduction. A rapidquench technique [3**] allowed measurement of CAMP levels in olfactory cilia preparations on a subsecond time scale. Less than 50ms after introduction of odorant, accumulation of CAMP reached a level four times higher than basal levels and the rate of accumulation was linearly related to ligand concentration. This CAMP increase was short-lived, and within approximately 3OOms, concentrations returned to pre-stimulated levels. This ‘pulse’ of cyclic nucleotide precedes the odorant-stimulated generator current [3**]. Two transduction
pathways
The odorants isomenthane and citralva, both sweet smelling odorant molecules, stimulate cAMP formation [3**,7**]. Pyrazine, a highly potent member of an odorant family classiiied as ‘putrid, failed to elicit cyclic nucleotide accumulation. Instead, an increase in IP3 production was observed. This increase in IP3 concentration, similar to that seen for other odorants and CAMP, was transient and preceded membrane depolarization [ 7**]. Earlier work has shown that odorants which efficiently stimulate adenylyl cyclase activity are often non-polar molecules. The more polar molecules such as pyrazine, though potent, minimally elevate CAMP generation in chemosensoty cilia. Addition of hydrocarbon side chains to the hydrophilic molecule (to decrease its polarity) resulted in increases in adenylyl cyclase activity and the
cascade
in olfactory
fruity/sweet quality pyrazine stimulates tion that odorants classes, each class tion pathway.
receptor
neurons
Bakalyar
and Reed
of the odorant [ 21. The discovery that IP3 production has led to the suggesmay be categorized into one of two activating a dilferent signal transduc-
Accumulation of the two types of second messengers appears to be dependent upon the involvement of two separate G proteins. Persistent activation of G, and G,-like a-subunits by cholera toxin, increased CAMP levels in rat olfactory cilia but did not affect IP3 generation. In contrast, pyrazine-activated IP3 generation was blocked by pertussis toxin [7**] which uncouples Gi and G, from the membrane bound receptors. These observations suggest that the CAMP olfactory cascade is mediated by a G,,like protein, G,r, and the IP3 second messenger pathway by a G,- or a G&e protein. As a result of the build up of distinct second messengers, the two odorant classes are likely to modulate the opening of different cation channels. The existence of cyclic nucleotide-gated cation channels in a variety of species has been described [ 151. Data obtained from whole-cell recordings of lobster olfactory receptor cells are suggestive of a heterogeneous population of odorant responsive channels which transport different ionic species [ I6*]. An IPj-gated Ca2+ -selective channel, in addition to a cAMP responsive non-specific cation channel, has been identified in catfish olfactory receptor neurons [17**].
Molecular olfactory
components in CAMP-mediated signal transduction
In recent years, molecular biological techniques have been used to dissect the CAMP second messenger cascade in olfactory receptor neurons. The olfactory system appears to use specialized variants of components expressed in non-sensory cells. The biochemical and electrophysiological characterization of some of these components have revealed properties that are important for their role in transduction of odorant stimuli. Golf The first discovered of the olfactory receptor neuron-specific proteins thought to be involved in the second messenger cascade, was the G,,-like protein, G,K,. In the neuroepithelium, G,u is the most abundant G protein capable of stimulating CAMP production [ 18*]. The protein has been specilically localized, using lmmunohistochemical techniques, to the sensory cilia [4], a location appropriate for the protein’s proposed role in olfactory signal transduction. Golf has been shown to stimulate adenylyl cyclase in a murine lymphoma cell line deficient in G,, [ 191. Only subtle differences in the activation properties of Golf and G, have been distinguished [ 191, leaving open the question as to why the olfactory system might have evolved a unique stimulatory G,-subunit. If specialization does not
205
206
Sensory systems
.
confer distinct biochemical properties, the presence of a separate gene for Goti may facilitate the expression of this stimulatory G protein at high levels in olfactory receptor neurons. Adenylyl
cyclase
Unlike G,K, an olfactory specilic adenylyl cyclase, termed type III, appears to be biochemically distinct from other characterized adenylyl cyclases. A mammalian cell line expressing the type III cDNA shows elevated adenylyl cyclase activity in response to forskolin, a diterpene capable of stimulating maximal adenylyl cyclase activity. The agent AlP4_ , which stimulates adenylyl cyclase via G protein activation, also brought about an increase in type III adenylyl cyclase activity in this system. The basal activity of the expressed type III adenylyl cyclase is not increased over background in this system. This contrasts with other cloned adenylyl cyclases, from brain and peripheral tissue, which when expressed in a similar manner produce high levels of CLAMPin the absence of forskolin or AlPb_ The exceedingly low basal, and high stimulated level of enzyme activity attributed to the type IIl protein may provide the olfactory sensory cilia, where the enzyme is abundant, with a large dynamic range for CAMP modulation. In the absence of odorant, low type III basal activ ity would maintain low cAMP levels. Upon odorant exposure, the activation of the adenylyl cyclase would quickly produce large increases in second messenger, switching the system from the ‘off state to ‘on’. This high signal-tonoise ratio could contribute to the exquisite sensitivity characteristic of the olfactory system [5-l. Channel
Messenger RNA encoding an olfactory-specific, cyclic nucleotide gated monovalent cation channel was shown to be expressed at high levels in the receptor neurons within the olfactory epithelium. As observed in patchclamp studies of the native channel [ 151, the cloned channel is gated by both cAMP and cGMP and is somewhat permeant to Ca* + . A Hill coefficient of 1.9 derived from a plot of current amplitude as a function of ligand concentration demonstrates cooperativity in olfactory channel opening. This suggests the channel may contribute to the overall cooperativity observed in odorant induced membrane depolarization [6**].
tible to secondary biotransformations. It is possible that these actions may serve a non-specific detoxification role, however, UGT,,, an olfactory-specific UDP glucuronosyl transferase localized to mucus secreting glands in the nasal epithelium, has been shown to preferentially conjugate hydroxylated odorants which activate the CAMP pathway [22**]. This modification reduces the odorants’ ability to stimulate adenylyl cyclase activity. The high cytochrome P450 enzyme activity noted in olfactory epithelium, may therefore be involved in a two step reaction of signal termination and odorant clearance.
Potential integration messenger cascades
of the separate
second
The observation that at least two second messenger signalling cascades are involved in olfaction raises the possibility that interaction between these pathways may play an important role in the propagation of the electrical signal to the brain. IP3 liberates Ca*+ from internal stores in some tissues and, in olfactory ciliaty membranes, it opens channels which permit an influx of extracellular Ca*+ [17-l. Calmodulin, a Ca*+ -dependent activator of many physiological processes, is abundant in frog sensory cilia and is competent to stimulate olfactory adenylyl cyclase activity in this species [&WI. The adenylyl cyclase activation by calmodulin is synergistically enhanced by GTP [8-l. Synergism implies that common elements in multiple pathways leading to odorant detection may exist. A Ca2+ -dependent calmodulinmediated regulation of CAMP levels points to dual regulation of adenylyl cyclase as a possible means of coupling the two pathways under certain conditions. A sufficiently large, odorant-induced IPj-mediated influx of Ca*+ into the receptor cilia may activate the adenylyl cyclase and the downstream components of the CAMP signalling path way. When expressed in human embryonic kidney cells, the type III olfactory-spectic adenylyl cyclase displayed a Ca*+/calmodulin-stimulation of CAMP production (HA Bakalyar and RR Reed, unpublished data). These preliminary data are consistent with observations in isolated frog cilia and provide a possible mechanism for interaction among the multiple olfactory signalling cascades.
Conclusion Signal terminalion
It seems likely that both intracellular and extracellular processes are important in the termination of the odorant signal. Phosphodiesterases degrade the intracellular second messenger. Additionally, hydrophobic odorants solubilized ln the mucus and lipid membrane must be prevented from further activating the signalling cascade. This may be accomplished by the concerted action of olfactory-speciiic cytochrome P450 mono-oxygenases [ 20*,21 l ] , and a UDP glucuronosyl transferase [21*,22-l. The P450 enzymes could serve to hydroxylate odorants causing them to become more hydrophilic and suscep-
Research in the past year has resulted in a greatly expanded understanding of the signal transduction mechanisms in olfactory receptor neurons. The kinetics of cAMP formation have been shown to be appropriate for mediation of odorant detection. The molecular cloning of components involved in this pathway has facilitated investigation into their biochemical properties. Previous studies on olfactory ciliary membranes had demonstrated that exposure to putrid molecules yielded only minute adenylyl cyclase responses with even the most potent odorants of this class. This year, IPJ has been strongly
The second
messenger
implicated as an alternative second messenger for the detection of putrid odorants. The identification of multiple pathways present in the sensory neurons suggests a common mechanism for all olfactory signalling. The interaction of independent second messenger cascades leads to attractive models for additional signal processing that may generate specificity at the biochemical and electrical level. The nature of these interactions and their importance in generating the information transmitted to the brain will be a major focus of research efforts in the coming year.
References
and recommended
Papers of special interest, published have been highlighted as: . of interest .. of outstanding interest 1.
GETCHEU
2.
SNYDER SH, SKLU
within
reading the annual
period
of review,
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BREEH H, BOEKI-IOFF 1, TARELLIS E: Rapid Kinetics of Second Messenger Formation in Olfactory Transduction. Nufure 199Q, 345:6S68. The kinetics of second messenger generation in mammals and insects are measured on a subsecrond time scale using a rapid quench technique. This paper demonstrates chat the concentrations of second messenger in the rat (CAMP) and in the cockroach (IPj) undergo a trans. ient and significant increase in response to odorant that precedes membrane depolarization. Previous studies have implicated these molecules in signalling cascades based on enzyme activity measurements. but until this paper, none had determined whether increased activity resulted in a significant build-up of second messenger that occurred with the kinetics appropriate for an agent with a causal role in membrane depolarization.
4.
JON& DT, REED RR: G,s an Olfactory Neuron Protein Involved in Odorant Signal Transduction. 1989, 244:79&795.
5. ..
BAKALYAR H&
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&ED RR: ldentiiication of a Specialized Adenylyl Cyclase that May Mediate Odorant Detection. Science 1990, 250:14031406. A cDNA encoding an adenylyl cyclase (type III), distinct from the brain form of the enzyme, was shown to be OlfaCtOI’y neuron specific. The type Ill protein was localized to the sensory cilia and could be activated by G, in a heterologous expression system suggesting a role in olfactory signal transduction. In these studies, type III adenyiyi cyclase exhibited a high activated rite of CAMP production and an exceedingIy low basaI activity, perhaps conferring the olfactory cilia with a large dynamic range of CAMP levels. DHAIUN
RS, YALI K-W,
SIZHRADER K4
REED RR Primary
SUUC-
ture and Functional Expression of a Cyclic NucleotideActivated Channel from Olfactory Neurons. Nulure 1990, 347:1&l-187. An olfactory neuron-specific message encoding a functional cation channel was isolated from a rat olfactory cDNA library. The cyclic nucleotide dependence of the cloned channel’s opening. as well as its permeation properties.correlated with observed properties of the native channel from toad sensory cilia [IS]. ..
7. ..
I, TAREIUIS E, STROT~IANN J, BREER H: Rapid Actiof Alternative Second Messenger Pathways in Olfac-
BOEKHOFF
vation
in olfactory
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tory Cilia from Rats by Diierent Odorants. &VI30 / 1!990, 924532458. This important paper demonstrates that IPJ in addition to the well studied CAMP pathway, operates as a second messenger 10 media@ mammalian odotant detection. The appearance of both IPJ and CAMP in response to odorants is transient and precedes membrane depoladzation. These two second messenger pathways are modulated by Werent G proteins and evoked in response to odorants representative of different molecular classes. 8. ..
RRH, RMXU AM: Olfactory Transduction: Cross-Talk Between Second-Messenger Systems. Biocbem~ 1990, 29:404WO54. This study was COmpleted before the IF’3 mediated pathway of O&tOly signal transduction was examined by Boekhoff et a.! [7*-l. A series of experiments examining the synergistic effecrs of Ca*+/calmodulin and GTP in frog olfactory ciliaty preparations led the authors to speculate that an unidentified reaction pathway leading fo an odor-induced increase in intracellular Ca*+ stimulates the action of adenyiyl cyclase in olfactory receptor neurons. ANHOLT
9. .
3. . .
6.
cascade
PRUNER J, HOLI V, SNOWMAN AM, SNYDER SH: Odorant-Binding Protein: Characterization of Ligand Binding. / Biol Ckm 1990, 265:61186125. This work represents a further attempt to characterize structu~ feaNreS of odoranr molecules and to correlate them with their binding affinity for odorant-binding protein. The most interesting information contained in the paper is a scatchard analysis of l&and binding to odor-ant-binding protein which suggests that the binding is negatively cooperative. 10.
B: Single Unit Recording for Olfactory Cilia. Biophys / 1’990, 57:109-1094. &se patch-clamp SNdieS on olfactory receptor neuronal cilia from frog were performed on neurons not dissociated from either the surrounding epithelial tissue or the mucosa. Using this method, odorant detection thresholds in the physiological range (PM) were obtained, in contrast to the large concentrations of odorant required fo elicit electrical response in single-unit patch-clamp sNdks on isolated neurons. FIUNGS S, LINDEMANN
11. .
AYYI~ C, PARANJAPE J, RODRIGUES V, SIDDIQI 0: Genetics of Olfactory Behavior in Drosophila Melanogaster. / Neurogen 1990, 6:243262. The isolation and characterization of six olfactory deficient mutant Lkcscpbih mekmogaster strains, some of which have been previousIy described in the LiteraNre, are presented. Mutations conferred tierential anosmias with a range of specilicities. These included a general unresponsiveness in a mutation allelic to the previously isolated ‘smellblind’ muration, a strain non- responsive when challenged with odorants of a particular chemical class, and highly specific mutant strains that were non-responsive to only certain odorants within a chemical class. 12.
REED RR How
Does
the
Nose
Know?
Cell 1990, 60:1-2.
kis brief review summarizes efforts in several systems fo examine the mechanism of odor detection and suggests possible models to account for the sensitivity and specificity of this sensory system. 13. ..
I AXEL R: A Novel Multigene Family May Encode Odorant Receptors: a Molecular Basis for Odor Recognition. Cell 1991, 65:17%187. Eighteen olfactory specific transcriptions of a large multigene family encoding proteins with the classical G-protein-coupled receptor Seven transmembrane domain StIUCNre were isolated from mt olfactory ep ithelium. A conservative estimation of the number of genes included in this family is calculated at lC0-200 members. The products of these genes may now be used to examine odorant binding properties, and their potential interaction with the second messenger cascade in olfactory signal transduction. 14. ..
BUCK
FIRESTEIN S, SHEPHERD GM, WERBUN FS: Tie Course of the Membrane Current Underlying Sensory Transduction in Salamander Olfactory Receptor Neurones. / Pm1 1990, 430:13>158. Pulses of odorant directed at sensory cilia of salamander olfactory receptor neurons, under whole-cell patch-clamp, generated current after approximately 500 ms concentration-independent latency. Current am-
207
208
Sensory
systems
plitude response to duration and concentration of the stimulus is consistent with the presence of a second messenger cascade in olfactory signal transduction. 15.
NAKAMURA T, GOLD
GH: A Cyclic
tance
Receptor
in Olfactory
Cilia.
Conduc1987, 325:442-@i4.
Nucleotide-Gated
N&we
16.
SCHMIEDEL-JAKOB
.
Whole Cell Recording Tom Lobster Olfactory Receptor Cells: Multiple Ionic Bases for the Receptor Potential. &cm senres 1990, 15:397-i05.
I,
MICHEL
WC,
ANDER?ON
PAV.
ACHE
BW:
Biochemical examination of two forms of G, (G, 5, G, t) and G,,lf in a murine cell line deficient in stimulatory G protein demonstrated a high degree of functional similarity between the three G protein a-subunits. Sensitiity to cholera toxin and GTP analogues w.s identical, and the three proteins demonstrated similar coupling interactions with activattd P-adrenergic receptor. 20. .
LURD ZLIPKO
D, K:
TAL
N.
Identification vel Olfactory-Specific curonosyl Transferase.
RL~INSTEIN
M,
KHHEN
M,
L~CET
D,
and Biochemical Analysis of NoCytochrome P-45OlIA and UDP-GluBitimlisfr)~ 1990, 29:7433-7&O.
Whole-cell current-clamp recordings on intact lobster olfactory neurons were used to assess the ion dependence of odor-induced olfactory receptor neuron potential. These studies identikd distinct neuronal populations exhibiting di5erent patterns of ionic requirements for odor-in. duced receptor potential.
Two integd membrane proteins enriched in de-ciliated hovine olfactory epithellum were purified electrophoretlcally and subjected fo partial peptide sequencing. .Secluence analysis revealed gp56 w-&5 highly homologous to UDP glucuronosyl trdnsfenw. and ~52 shared a high degree of similarity to proteins in rhe qqochrome PrSOIlA famib.
17. ..
21.
NEF
.
Specilic Cy-tochrome P-450 (P45Oolfl; ture and Developmental Regulation.
RESTREPO D,
MNAMOTO
uli Trigger Influx Channel Cat&h.
T, BRYN
Odor StimNeurons of the
BP, TEFTER JH:
of Calcium into Olfactory Science 1990, 249~11661168.
In catfish, Ca2+ external to olfactory receptor neurons proved necessary for the electro-olfactogram, the odorant.induced increase in internal Ca2+, and IF’3 induction of membrane depolarization in olfactory receptor neurons. Ciliaty membrane vesicles fused into lipid bilayers led to the biochemical identification of an IPj.gated Ca’+ channel on the plasma membrane of catfish olfactory receptor neurons. 18.
.
Distribution of the Stimulatory GTP-Binding teins, G, and Golf Within Olfactory Neuroepithelium senses 1990, 15:333340.
JONES DT:
Pro&em
Olfactory ciliary protein, normal epithelial and neuromdepleted epithe. lial protein extracts were examined by western analysis to determine the relative abundance of G, and Golf a-subunits in the neuroepithelium. The results confirmed previous data obtained at the messenger RNA level, that Go8 was by far the most abundant G protein in olfac. tory sensoty cilia. 19.
.
JONES DT, MAVES cal Characterization teins. J Bid Cbem
SB, BOURNE m of Three Stimulatory 1990, 265:2671-2676.
RR: BiochemiGTP-Binding Pro-
REED
P,
L\RADEE
TM,
KAGlh!OTO
K,
MEYER
IIGl): .I Bid
LIA
OlfactoryGene Struc-
Chmn 1990, 265290325Xl7. P450olf1, an olfactoryspecific qtochrome mono-oxzgenase, is en. coded on a single copy gene that was shown to be structurally simi. lar to other genes of the P450IIG family. The appearance of PrSOolfI coincided with the onxt of a postnatal increase in olfactory acuiv. 22.
LAZARD D, ZLIPKO
..
KHEN M,
UDP
K. POR~A Y. NEF P. LG!!o\~
~ANCET D: Odorant Signal Termination Glucuronosyl Transferax. Nahtre 1990,
J. HORN
S,
by Olfactory 349:79C-793.
A cDNA clone encoding a protein with UDP glucuronosg tramferase activity wds isolated from a rat olfactory cDNA library. The olfactoty3pecific encoded protein formed glucuronos)~ conjugates with some hydroxyiated odorant molecules. This moditication of odorant molecules diminished their abiliv to stimulate adenylyl q&se.
HA Bwar and RR Reed, Howard Hughes Medical Institute, Depanment of Molecular Biology and Genetics, The Johns Hopkins School of Medicine, 725 North Wolfe Street, Baltimore. Maryland 21205, USA
receptor
neurons Heather Howard
Hughes
Medical
A. Bakalyar
Institute,
and Randall
The Johns Hopkins
School
R. Reed
of Medicine,
Baltimore,
Maryland,
USA
The molecular cloning of components involved in the CAMP second messenger cascade has allowed their biochemical characterization and revealed properties that are important for their role in sensory transduction. Recent evidence suggests inositol 1,4,5-trisphosphate functions as an additional second messenger in olfactory signalling. The interaction of these two pathways may contribute to the sensitivity of the olfactory system. Current
Opinion
in Neurobiology
tribute to the overall sensitivity and specificity associated with odor detection.
Introduction Olfaction is the process by which information
regarding the external chemical environment is transduced into an electro-chemical signalling cascade which leads to an action potential encoding for the identity and intensity of the original stimulus. In mammals, odorants (compounds which can be perceived) are drawn through the nasal lumen, solubilized and brought into direct contact with the olfactory receptor neurons. Long, non-motile cilia on the apical membrane of these bipolar neurons project into the mucus layer which bathes the surfa* of the olfactoIy epithelium. These ciliary structures, exposed to the external environment, are the site of the primary olfactory events: odorant recognition, transduction of the external stimulus into an internal chemical signal and subsequent membrane depolarization [ 11. Membrane depolarization initiates an action potential which propagates the signal to second order neurons in a specialized processing region of the brain, the olfactory bulb. Psychophysical studies have demonstrated that the human olfactory system is able to discriminate between more than 10 000 odorants with high sensitivity, some airborne molecules can be detected at concentrations as low as a few parts per trillion
[21. The sensitivity attributed to this system is at least in part achieved by a signal-amplifying second messenger cascade. A GTP-dependent increase in adenylyl cyclase activity in response to ‘fruity’ odorants like citralva is obsend in olfactory cilia. This increased enzyme activity results in a transient rise in CAMP concentration that precedes odorant-induced membrane depolarization [ 3~=]. Several components of classical GTP-binding protein (G protein) signalling cascades (a G, subunit of the G protein heterotrimer, an adenyiyl cyclase, and an ion channel gated by a cyclic nucleotide) appear to have evolved specialized counterparts in the olfactory system [4,5**,ti*]. Functional interaction of these components may con-
C protein---GTP-binding
204
1991, 1:204-208
@ Current
Recently, Breer’s laboratory [7**] has demonstrated that the odorant pyrazine produces an intracellular rise in a different second messenger, inositol 1,4,5-&phosphate (IP$, in rat sensory cilia. This implies that a second messenger pathway other than CAMP, responsive to different odorant stimuli, may be involved in mammalian olfaction. The mechanism by which these separate second messenger olfactory cascades may be integrated in the complete olfactory process is as yet unknown, although a role for Ca2 + -activated calmodulin has been hypothesized [ W]
Odorant interaction generation
to second
messenger
In mammals, airborne odorants must diffuse into the mucus layer bathing the nasal epithelium prior to contact with recognition sites on the olfactory neuronal cilia. Odorant-binding proteins may play a role in concentrating some odorants and in presenting odorant molecules to the recognition sites. The observed negative cooperative binding of odorant ligand to odomnt-binding protein may facilitate the uptake of odorant from ambient air in the nasal lumen and its subsequent release in the mucus layer (9*]. Loose patch-clamp recording studies on in situ olfactory receptor neurons indicate that ionic conductances appear in response to picomolar concentrations of applied odorant [lo*]. Once the odorant has reached the olfactory receptor neuron surface, interaction with the recognition site, presumably a protein receptor molecule, takes place. The mechanism conferring specificity to this interaction has yet to be elucidated; a large number of highly specific receptors may be involved, or a smaller population of unique recep-
Abbreviations protein; IP3-inositol Biology
prior
1,4,5-trisphosphate.
Ltd ISSN 09594388
The second
messenger
tom with broadly overlapping specificities may act combinatorially to discriminate chemically distinct odorants [ 1,11*,12*]. The recently described multigene family encoding olfactory-specific, seven transmembrane domain proteins may encode the repertoire of putative odorant receptors [ 13**]. These molecules may prove useful for investigating the origin of specilicity in odorant detection.
The mechanisms generation
of second
messenger
Kinetics
Firestein et al. [ 14.01 have employed a whole-cell patchclamp technique to delineate the time course of the salamander olfactory neuron response to odorant. The application of odorant pulses, directed at the cilia, elicited a current after the concentration-independent latency penod of approximately 500 ms. The magnitude of the current response was a function of both stimulus duration and odorant concentration over a dynamic concentration range of approximately one order of magnitude. Short odorant pulses (less than 1OOm.s) generated a current that peaked after odorant had dilfused to negligible lev els. These observations are consistent with the presence of an integrating step, for instance, the generation of second messenger, in ciliary odorant-induced membrane depolarization [ 14**]. Modulation of cAMP concentrations in rat olfactory cilia was shown to follow the kinetics expected for such a second messenger in olfactory signal transduction. A rapidquench technique [3**] allowed measurement of CAMP levels in olfactory cilia preparations on a subsecond time scale. Less than 50ms after introduction of odorant, accumulation of CAMP reached a level four times higher than basal levels and the rate of accumulation was linearly related to ligand concentration. This CAMP increase was short-lived, and within approximately 3OOms, concentrations returned to pre-stimulated levels. This ‘pulse’ of cyclic nucleotide precedes the odorant-stimulated generator current [3**]. Two transduction
pathways
The odorants isomenthane and citralva, both sweet smelling odorant molecules, stimulate cAMP formation [3**,7**]. Pyrazine, a highly potent member of an odorant family classiiied as ‘putrid, failed to elicit cyclic nucleotide accumulation. Instead, an increase in IP3 production was observed. This increase in IP3 concentration, similar to that seen for other odorants and CAMP, was transient and preceded membrane depolarization [ 7**]. Earlier work has shown that odorants which efficiently stimulate adenylyl cyclase activity are often non-polar molecules. The more polar molecules such as pyrazine, though potent, minimally elevate CAMP generation in chemosensoty cilia. Addition of hydrocarbon side chains to the hydrophilic molecule (to decrease its polarity) resulted in increases in adenylyl cyclase activity and the
cascade
in olfactory
fruity/sweet quality pyrazine stimulates tion that odorants classes, each class tion pathway.
receptor
neurons
Bakalyar
and Reed
of the odorant [ 21. The discovery that IP3 production has led to the suggesmay be categorized into one of two activating a dilferent signal transduc-
Accumulation of the two types of second messengers appears to be dependent upon the involvement of two separate G proteins. Persistent activation of G, and G,-like a-subunits by cholera toxin, increased CAMP levels in rat olfactory cilia but did not affect IP3 generation. In contrast, pyrazine-activated IP3 generation was blocked by pertussis toxin [7**] which uncouples Gi and G, from the membrane bound receptors. These observations suggest that the CAMP olfactory cascade is mediated by a G,,like protein, G,r, and the IP3 second messenger pathway by a G,- or a G&e protein. As a result of the build up of distinct second messengers, the two odorant classes are likely to modulate the opening of different cation channels. The existence of cyclic nucleotide-gated cation channels in a variety of species has been described [ 151. Data obtained from whole-cell recordings of lobster olfactory receptor cells are suggestive of a heterogeneous population of odorant responsive channels which transport different ionic species [ I6*]. An IPj-gated Ca2+ -selective channel, in addition to a cAMP responsive non-specific cation channel, has been identified in catfish olfactory receptor neurons [17**].
Molecular olfactory
components in CAMP-mediated signal transduction
In recent years, molecular biological techniques have been used to dissect the CAMP second messenger cascade in olfactory receptor neurons. The olfactory system appears to use specialized variants of components expressed in non-sensory cells. The biochemical and electrophysiological characterization of some of these components have revealed properties that are important for their role in transduction of odorant stimuli. Golf The first discovered of the olfactory receptor neuron-specific proteins thought to be involved in the second messenger cascade, was the G,,-like protein, G,K,. In the neuroepithelium, G,u is the most abundant G protein capable of stimulating CAMP production [ 18*]. The protein has been specilically localized, using lmmunohistochemical techniques, to the sensory cilia [4], a location appropriate for the protein’s proposed role in olfactory signal transduction. Golf has been shown to stimulate adenylyl cyclase in a murine lymphoma cell line deficient in G,, [ 191. Only subtle differences in the activation properties of Golf and G, have been distinguished [ 191, leaving open the question as to why the olfactory system might have evolved a unique stimulatory G,-subunit. If specialization does not
205
206
Sensory systems
.
confer distinct biochemical properties, the presence of a separate gene for Goti may facilitate the expression of this stimulatory G protein at high levels in olfactory receptor neurons. Adenylyl
cyclase
Unlike G,K, an olfactory specilic adenylyl cyclase, termed type III, appears to be biochemically distinct from other characterized adenylyl cyclases. A mammalian cell line expressing the type III cDNA shows elevated adenylyl cyclase activity in response to forskolin, a diterpene capable of stimulating maximal adenylyl cyclase activity. The agent AlP4_ , which stimulates adenylyl cyclase via G protein activation, also brought about an increase in type III adenylyl cyclase activity in this system. The basal activity of the expressed type III adenylyl cyclase is not increased over background in this system. This contrasts with other cloned adenylyl cyclases, from brain and peripheral tissue, which when expressed in a similar manner produce high levels of CLAMPin the absence of forskolin or AlPb_ The exceedingly low basal, and high stimulated level of enzyme activity attributed to the type IIl protein may provide the olfactory sensory cilia, where the enzyme is abundant, with a large dynamic range for CAMP modulation. In the absence of odorant, low type III basal activ ity would maintain low cAMP levels. Upon odorant exposure, the activation of the adenylyl cyclase would quickly produce large increases in second messenger, switching the system from the ‘off state to ‘on’. This high signal-tonoise ratio could contribute to the exquisite sensitivity characteristic of the olfactory system [5-l. Channel
Messenger RNA encoding an olfactory-specific, cyclic nucleotide gated monovalent cation channel was shown to be expressed at high levels in the receptor neurons within the olfactory epithelium. As observed in patchclamp studies of the native channel [ 151, the cloned channel is gated by both cAMP and cGMP and is somewhat permeant to Ca* + . A Hill coefficient of 1.9 derived from a plot of current amplitude as a function of ligand concentration demonstrates cooperativity in olfactory channel opening. This suggests the channel may contribute to the overall cooperativity observed in odorant induced membrane depolarization [6**].
tible to secondary biotransformations. It is possible that these actions may serve a non-specific detoxification role, however, UGT,,, an olfactory-specific UDP glucuronosyl transferase localized to mucus secreting glands in the nasal epithelium, has been shown to preferentially conjugate hydroxylated odorants which activate the CAMP pathway [22**]. This modification reduces the odorants’ ability to stimulate adenylyl cyclase activity. The high cytochrome P450 enzyme activity noted in olfactory epithelium, may therefore be involved in a two step reaction of signal termination and odorant clearance.
Potential integration messenger cascades
of the separate
second
The observation that at least two second messenger signalling cascades are involved in olfaction raises the possibility that interaction between these pathways may play an important role in the propagation of the electrical signal to the brain. IP3 liberates Ca*+ from internal stores in some tissues and, in olfactory ciliaty membranes, it opens channels which permit an influx of extracellular Ca*+ [17-l. Calmodulin, a Ca*+ -dependent activator of many physiological processes, is abundant in frog sensory cilia and is competent to stimulate olfactory adenylyl cyclase activity in this species [&WI. The adenylyl cyclase activation by calmodulin is synergistically enhanced by GTP [8-l. Synergism implies that common elements in multiple pathways leading to odorant detection may exist. A Ca2+ -dependent calmodulinmediated regulation of CAMP levels points to dual regulation of adenylyl cyclase as a possible means of coupling the two pathways under certain conditions. A sufficiently large, odorant-induced IPj-mediated influx of Ca*+ into the receptor cilia may activate the adenylyl cyclase and the downstream components of the CAMP signalling path way. When expressed in human embryonic kidney cells, the type III olfactory-spectic adenylyl cyclase displayed a Ca*+/calmodulin-stimulation of CAMP production (HA Bakalyar and RR Reed, unpublished data). These preliminary data are consistent with observations in isolated frog cilia and provide a possible mechanism for interaction among the multiple olfactory signalling cascades.
Conclusion Signal terminalion
It seems likely that both intracellular and extracellular processes are important in the termination of the odorant signal. Phosphodiesterases degrade the intracellular second messenger. Additionally, hydrophobic odorants solubilized ln the mucus and lipid membrane must be prevented from further activating the signalling cascade. This may be accomplished by the concerted action of olfactory-speciiic cytochrome P450 mono-oxygenases [ 20*,21 l ] , and a UDP glucuronosyl transferase [21*,22-l. The P450 enzymes could serve to hydroxylate odorants causing them to become more hydrophilic and suscep-
Research in the past year has resulted in a greatly expanded understanding of the signal transduction mechanisms in olfactory receptor neurons. The kinetics of cAMP formation have been shown to be appropriate for mediation of odorant detection. The molecular cloning of components involved in this pathway has facilitated investigation into their biochemical properties. Previous studies on olfactory ciliary membranes had demonstrated that exposure to putrid molecules yielded only minute adenylyl cyclase responses with even the most potent odorants of this class. This year, IPJ has been strongly
The second
messenger
implicated as an alternative second messenger for the detection of putrid odorants. The identification of multiple pathways present in the sensory neurons suggests a common mechanism for all olfactory signalling. The interaction of independent second messenger cascades leads to attractive models for additional signal processing that may generate specificity at the biochemical and electrical level. The nature of these interactions and their importance in generating the information transmitted to the brain will be a major focus of research efforts in the coming year.
References
and recommended
Papers of special interest, published have been highlighted as: . of interest .. of outstanding interest 1.
GETCHEU
2.
SNYDER SH, SKLU
within
reading the annual
period
of review,
TV, GETCHIXI. ML Peripheral Mechanisms of Offiction: Biochemistry and Neurophysiology. In Neurobiology of Taste and Stnell, edited by Finger TE and Silver WK [book]. New York: Wiley Interscience 1987, Chapter 5, pp 91-123.
PB, PEVSNER J: Olfactory Receptor Mechanisms, Odorant-Binding Protein and Adenylyl Cyclase. In Molecular Neumbiolog)~ o/ the OlJkctoq~ Qskm7 edited by Margolis FL and Getchell TV [book]. New York: Plenum Publishing Corporation 1988, pp 324.
BREEH H, BOEKI-IOFF 1, TARELLIS E: Rapid Kinetics of Second Messenger Formation in Olfactory Transduction. Nufure 199Q, 345:6S68. The kinetics of second messenger generation in mammals and insects are measured on a subsecrond time scale using a rapid quench technique. This paper demonstrates chat the concentrations of second messenger in the rat (CAMP) and in the cockroach (IPj) undergo a trans. ient and significant increase in response to odorant that precedes membrane depolarization. Previous studies have implicated these molecules in signalling cascades based on enzyme activity measurements. but until this paper, none had determined whether increased activity resulted in a significant build-up of second messenger that occurred with the kinetics appropriate for an agent with a causal role in membrane depolarization.
4.
JON& DT, REED RR: G,s an Olfactory Neuron Protein Involved in Odorant Signal Transduction. 1989, 244:79&795.
5. ..
BAKALYAR H&
Specific-G Science
&ED RR: ldentiiication of a Specialized Adenylyl Cyclase that May Mediate Odorant Detection. Science 1990, 250:14031406. A cDNA encoding an adenylyl cyclase (type III), distinct from the brain form of the enzyme, was shown to be OlfaCtOI’y neuron specific. The type Ill protein was localized to the sensory cilia and could be activated by G, in a heterologous expression system suggesting a role in olfactory signal transduction. In these studies, type III adenyiyi cyclase exhibited a high activated rite of CAMP production and an exceedingIy low basaI activity, perhaps conferring the olfactory cilia with a large dynamic range of CAMP levels. DHAIUN
RS, YALI K-W,
SIZHRADER K4
REED RR Primary
SUUC-
ture and Functional Expression of a Cyclic NucleotideActivated Channel from Olfactory Neurons. Nulure 1990, 347:1&l-187. An olfactory neuron-specific message encoding a functional cation channel was isolated from a rat olfactory cDNA library. The cyclic nucleotide dependence of the cloned channel’s opening. as well as its permeation properties.correlated with observed properties of the native channel from toad sensory cilia [IS]. ..
7. ..
I, TAREIUIS E, STROT~IANN J, BREER H: Rapid Actiof Alternative Second Messenger Pathways in Olfac-
BOEKHOFF
vation
in olfactory
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Bakalyar and Reed
tory Cilia from Rats by Diierent Odorants. &VI30 / 1!990, 924532458. This important paper demonstrates that IPJ in addition to the well studied CAMP pathway, operates as a second messenger 10 media@ mammalian odotant detection. The appearance of both IPJ and CAMP in response to odorants is transient and precedes membrane depoladzation. These two second messenger pathways are modulated by Werent G proteins and evoked in response to odorants representative of different molecular classes. 8. ..
RRH, RMXU AM: Olfactory Transduction: Cross-Talk Between Second-Messenger Systems. Biocbem~ 1990, 29:404WO54. This study was COmpleted before the IF’3 mediated pathway of O&tOly signal transduction was examined by Boekhoff et a.! [7*-l. A series of experiments examining the synergistic effecrs of Ca*+/calmodulin and GTP in frog olfactory ciliaty preparations led the authors to speculate that an unidentified reaction pathway leading fo an odor-induced increase in intracellular Ca*+ stimulates the action of adenyiyl cyclase in olfactory receptor neurons. ANHOLT
9. .
3. . .
6.
cascade
PRUNER J, HOLI V, SNOWMAN AM, SNYDER SH: Odorant-Binding Protein: Characterization of Ligand Binding. / Biol Ckm 1990, 265:61186125. This work represents a further attempt to characterize structu~ feaNreS of odoranr molecules and to correlate them with their binding affinity for odorant-binding protein. The most interesting information contained in the paper is a scatchard analysis of l&and binding to odor-ant-binding protein which suggests that the binding is negatively cooperative. 10.
B: Single Unit Recording for Olfactory Cilia. Biophys / 1’990, 57:109-1094. &se patch-clamp SNdieS on olfactory receptor neuronal cilia from frog were performed on neurons not dissociated from either the surrounding epithelial tissue or the mucosa. Using this method, odorant detection thresholds in the physiological range (PM) were obtained, in contrast to the large concentrations of odorant required fo elicit electrical response in single-unit patch-clamp sNdks on isolated neurons. FIUNGS S, LINDEMANN
11. .
AYYI~ C, PARANJAPE J, RODRIGUES V, SIDDIQI 0: Genetics of Olfactory Behavior in Drosophila Melanogaster. / Neurogen 1990, 6:243262. The isolation and characterization of six olfactory deficient mutant Lkcscpbih mekmogaster strains, some of which have been previousIy described in the LiteraNre, are presented. Mutations conferred tierential anosmias with a range of specilicities. These included a general unresponsiveness in a mutation allelic to the previously isolated ‘smellblind’ muration, a strain non- responsive when challenged with odorants of a particular chemical class, and highly specific mutant strains that were non-responsive to only certain odorants within a chemical class. 12.
REED RR How
Does
the
Nose
Know?
Cell 1990, 60:1-2.
kis brief review summarizes efforts in several systems fo examine the mechanism of odor detection and suggests possible models to account for the sensitivity and specificity of this sensory system. 13. ..
I AXEL R: A Novel Multigene Family May Encode Odorant Receptors: a Molecular Basis for Odor Recognition. Cell 1991, 65:17%187. Eighteen olfactory specific transcriptions of a large multigene family encoding proteins with the classical G-protein-coupled receptor Seven transmembrane domain StIUCNre were isolated from mt olfactory ep ithelium. A conservative estimation of the number of genes included in this family is calculated at lC0-200 members. The products of these genes may now be used to examine odorant binding properties, and their potential interaction with the second messenger cascade in olfactory signal transduction. 14. ..
BUCK
FIRESTEIN S, SHEPHERD GM, WERBUN FS: Tie Course of the Membrane Current Underlying Sensory Transduction in Salamander Olfactory Receptor Neurones. / Pm1 1990, 430:13>158. Pulses of odorant directed at sensory cilia of salamander olfactory receptor neurons, under whole-cell patch-clamp, generated current after approximately 500 ms concentration-independent latency. Current am-
207
208
Sensory
systems
plitude response to duration and concentration of the stimulus is consistent with the presence of a second messenger cascade in olfactory signal transduction. 15.
NAKAMURA T, GOLD
GH: A Cyclic
tance
Receptor
in Olfactory
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Conduc1987, 325:442-@i4.
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16.
SCHMIEDEL-JAKOB
.
Whole Cell Recording Tom Lobster Olfactory Receptor Cells: Multiple Ionic Bases for the Receptor Potential. &cm senres 1990, 15:397-i05.
I,
MICHEL
WC,
ANDER?ON
PAV.
ACHE
BW:
Biochemical examination of two forms of G, (G, 5, G, t) and G,,lf in a murine cell line deficient in stimulatory G protein demonstrated a high degree of functional similarity between the three G protein a-subunits. Sensitiity to cholera toxin and GTP analogues w.s identical, and the three proteins demonstrated similar coupling interactions with activattd P-adrenergic receptor. 20. .
LURD ZLIPKO
D, K:
TAL
N.
Identification vel Olfactory-Specific curonosyl Transferase.
RL~INSTEIN
M,
KHHEN
M,
L~CET
D,
and Biochemical Analysis of NoCytochrome P-45OlIA and UDP-GluBitimlisfr)~ 1990, 29:7433-7&O.
Whole-cell current-clamp recordings on intact lobster olfactory neurons were used to assess the ion dependence of odor-induced olfactory receptor neuron potential. These studies identikd distinct neuronal populations exhibiting di5erent patterns of ionic requirements for odor-in. duced receptor potential.
Two integd membrane proteins enriched in de-ciliated hovine olfactory epithellum were purified electrophoretlcally and subjected fo partial peptide sequencing. .Secluence analysis revealed gp56 w-&5 highly homologous to UDP glucuronosyl trdnsfenw. and ~52 shared a high degree of similarity to proteins in rhe qqochrome PrSOIlA famib.
17. ..
21.
NEF
.
Specilic Cy-tochrome P-450 (P45Oolfl; ture and Developmental Regulation.
RESTREPO D,
MNAMOTO
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T, BRYN
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BP, TEFTER JH:
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In catfish, Ca2+ external to olfactory receptor neurons proved necessary for the electro-olfactogram, the odorant.induced increase in internal Ca2+, and IF’3 induction of membrane depolarization in olfactory receptor neurons. Ciliaty membrane vesicles fused into lipid bilayers led to the biochemical identification of an IPj.gated Ca’+ channel on the plasma membrane of catfish olfactory receptor neurons. 18.
.
Distribution of the Stimulatory GTP-Binding teins, G, and Golf Within Olfactory Neuroepithelium senses 1990, 15:333340.
JONES DT:
Pro&em
Olfactory ciliary protein, normal epithelial and neuromdepleted epithe. lial protein extracts were examined by western analysis to determine the relative abundance of G, and Golf a-subunits in the neuroepithelium. The results confirmed previous data obtained at the messenger RNA level, that Go8 was by far the most abundant G protein in olfac. tory sensoty cilia. 19.
.
JONES DT, MAVES cal Characterization teins. J Bid Cbem
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RR: BiochemiGTP-Binding Pro-
REED
P,
L\RADEE
TM,
KAGlh!OTO
K,
MEYER
IIGl): .I Bid
LIA
OlfactoryGene Struc-
Chmn 1990, 265290325Xl7. P450olf1, an olfactoryspecific qtochrome mono-oxzgenase, is en. coded on a single copy gene that was shown to be structurally simi. lar to other genes of the P450IIG family. The appearance of PrSOolfI coincided with the onxt of a postnatal increase in olfactory acuiv. 22.
LAZARD D, ZLIPKO
..
KHEN M,
UDP
K. POR~A Y. NEF P. LG!!o\~
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S,
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A cDNA clone encoding a protein with UDP glucuronosg tramferase activity wds isolated from a rat olfactory cDNA library. The olfactoty3pecific encoded protein formed glucuronos)~ conjugates with some hydroxyiated odorant molecules. This moditication of odorant molecules diminished their abiliv to stimulate adenylyl q&se.
HA Bwar and RR Reed, Howard Hughes Medical Institute, Depanment of Molecular Biology and Genetics, The Johns Hopkins School of Medicine, 725 North Wolfe Street, Baltimore. Maryland 21205, USA