CURRENT AWARENES

The mysteries of sigma receptors: but also from identical transmern-

brane topologies (Fig. lb). The
new family members reveal a role in sigma, receptor and the fungal ERG2
proteins (cloned from S~ccharonzytcs
cholesterol synthesis cerevisiae, Magnaporthe grisen and
Ustilago may&s) have in common ~II
aminoterminal membrane anchor
Fabian F. Moebius, Jiirg Striessnig and Hartmut Glossmann
and two additional stretches of
hydrophobic residues involved in
In the past decade, extensive research the sigma, receptor and the dis- substrate binding (see Ref. 11 and
has been performed on sigma recep- covery of two new members of the references therein). Despite this in-
tors in the brain and other (e.g. sigma receptor family, strongly sug- timate structural relationship the
endocrine) tissue+. These recep- gest a role for these binding proteins function of the sigma, receptor is
tors are believed to mediate the in postsqualene sterol biosynthesis. not yet known. In contrast to the
immunosuppressant, antipsychotic The implications of these findings emopamil binding protein (EBP)
and neuroprotective effects elicited for the interpretation of the biologi- (see overleaf), sigma, receptors
by sigma ligands such as haloperi- cal effects of sigma ligands and their failed to complement isomerization-
dol, ditolylguanidine and penta- potential use in drug development deficient yeast mutants. It is
zocine4. Since endogenous agonists are discussed below. unknown whether the yeast ERG2
are not known (progesterone is a protein exhibits isomerase activity in
possible but controversial candi- The sigma, receptor: a mammalian cells or whether addi-
date)s,6, sigma sites cannot be classi- mammalian homologue of the tional cofactors are needed.
fied as receptors in the sense of being yeast sterol C,-C, isomerase
agonist-activated mediators of sig- The high-affinity sigma, receptor The yeast sterol C,-C,
nal transduction. Although their probes pentazocineg and azidopamil isomerase: an ancestral sigma
preferential localization in the mem- (an arylazide photoaffinity label) receptor
brane of the endoplasmic reticulum allowed the isolation, amino acid The structural similarity between
is different from neurotransmitter sequencing and cDNA cloning of the sigma, receptor and yeast iso-
receptors, the widely used term the sigma, receptorll. Heterologous merase suggested that the ERG2
sigma receptor will be maintained expression unequivocally proves protein might also carry a high-
throughout this article. that the isolated protein has all the affinity binding domain for sigma
Earlier studies suggested a binding properties of brain sigma ligands. Indeed, a high affinity
number of roles for sigma receptors. receptors. Its amino acid sequence haloperidol binding site (K, 0.5~)
They were thought to represent shows no homology to known mam- was found to be associated with the
I450 isoenzymes, or part of the malian proteins but shares 30% iden- ERG2 protein of yeast;. The ERG2
IP, [inositol(l,4,5)-trisphosphate] tity with the ERG2 gene product of protein also displayed high affinity
signalling cascade. One study yeast (Fig. la). This gene encodes a for other sigma ligands such as
showed them to be targets of intra- sterol C,-C, isomerase of the ergo- opipramol, ifenprodil, amiodarone
cellular endogenous ligands re- sterol biosynthetic pathway cata- and emopamil (Table 1). Therefore,
leased in a Ca2+-dependent manner. lysing a shift of the CB(9) double the sterol C,-C, isomerase of yeast
However, their biological function bond in the B-ring of sterols to the seems to represent an ancestral
remained enigmatic because no C7(8) position. Yeast strains with member of the sigma receptor fam-
specific tools such as antibodies, ERG2 mutations only grow in the ily3. Sigma ligands such as SR31747,
DNA probes and recombinant presence of exogenous ergosterol haloperidol and ifenprodil inhibit
receptors were available. As a conse- because ergosterol precursors can- the growth of yeast. This antiprolif- F. F. Moebius.
quence sigma receptors, originally not substitute for mature ergosterol erative action is mediated bv inhibi- Senior Research
postulated to explain the psycho- unless the strains carry additional tion of the catalytic activity of the Assaclate,
J. Striessnig,
mimetic effects of SKF10047 (Ref. 7), mutationslz. In plants and mammals sterol C,-C, isomerase due to inter- AssIstant Professor
were solely defined as high-affinity the isomerization is also required for action with the sigma-drug binding and Section Chief
haloperidol-sensitive binding sites, the action of a delta7-reductase satu- site of the enzymel2,. Yeast sterol Molecular
Pharmacology,
unrelated to opioid and N-methyl-D- rating the C7(8) double bond (Fig. 2). biosynthesis mutants helped to and
aspartate (NMDA) receptors*. The The isomerase is therefore required reveal the ergosterol requirement of H. Glossmann.
classification of two subtypes* could for the de nova synthesis of choles- many cellular functions14. S. CCYE- ChaIrman,
Insmut fljr
not solve the sigma enigma? what is terol in humans. zlisiae, a well characterized unicellu- Blochemische
the structure and biological function The structural similarity of the lar organism, therefore makes an Pharmakologle.
of these sites and how do they medi- sigma, receptor to the ERG2 protein Urwersitdt Innsbruck
excellent model to study biochemi-
Peter Mayr StraRe 1,
ate the pharmacological effects of is not only obvious from the amino cal effects of sigma ligands, e.g. on A-6020 Innsbruck.
sigma ligands? The recent cloning of acid sequence alignment (Fig. la) membrane permeability. Austria

t lYY7, Ilsevier Science Ltd Pll: SO165-6137(Y~301~1~7-1 TIPS March lYY7 (Vnl. 1X) 67
 CURRENT AWARENESS

a GP 1 MQWAVGRRWLWVALFLAAVAVLTQIVWLWLGTQNFVFQREEIAQLARQYA The mammalian sterol C,C,

.. . I:::.): : .::: : . I::I:...:.::.. isomerase: another member of
SC 1 .. . . ..MKFFPLLLLIGVVGYIMNVLFTTWLPTNYMFDPKTLNEICNSVI
".. .
the sigma receptor family
GP 51 G...LDHELAFSKLIVELR.....RLHPHVLPDEELQWVFVNAGGWMGAM We recently characterized, iso-
: ..: I...l:::I :....:. .:lIl/Ill Il.!
SC 45 SKHNAAEGLSTEDLLQDVRDALASHYDEYINRYVKEEWVFNNAGGAMGQM
lated and cloned a high-affinity
binding protein, known as
GP 93 CLLHASLSEYVLLFGTALGSPRHSGRYWAEISDTIISGTFHQWREGTTSE
.:lII/:/I/::IIllI:/.. I,/ .:I: II: II . I..1
emopamil binding protein (EBP)
SC 95 IILHASVSEYLILFGTAVGTEGHTGVHFADDYFTILHGTQIAALPYTEAE which binds the anti-ischaemic Cal+
GP 143 VFYPGETVVHGPGEATAVEWGPNTWMVEYGRGVIPSTLGFALADTVFSTQ
channel antagonist emopamil, and
l:lI/ .I).......: :I.:./ I I: I./:: I I. 11 other experimental neuroprotec-
SC 145 VYTPGMTHHLKKGYAKQYSMPGGSFALELAQGWIPCMLPFGFLDTFSSTL tants (e.g. ifenprodil)r5-18. Surpris-
GP 193 DFLTLFYTLRVYARAL.QLLTTYLFGQDP ingly, this unique protein shared
I:.\\: I:: Il.:.\/. I pharmacological similarities with
SC 195 DLYTLYRTVYLTARDMKNLLQNKKF
I
sigma receptors (Table 1). Loison
b 3t 'T' and co-workers discovered that the
EBP is also a sterol Cs-C, isomerase
and, thus, the mammalian counter-
part of the yeast ERG2 protein.
This implied that, in addition to
pharmacology, this catalytic activity
is a common denominator of sigma
receptors of yeast and mammals.
100 150 Strikingly, neither the amino acid
Amino acid residue
sequence19 nor the transmembrane
topology (Fig. lc) of yeast and
human sterol C,-C, isomerases are
related. Whereas the ERG2 protein
is anchored in the membrane of
the endoplasmic reticulum by an
aminoterminal a-helix the EBP has
four transmembrane segments (Fig.
lc,d). The existence of a sterol C,-C,
100 150
Amino acid residue isomerase in mammals that is struc-
turally unrelated to the yeast
d enzyme suggests that the two
enzymes evolved independently.
Most interestingly, the sterol C,-C,
isomerase of the plant Arubidopsis
cytoplasm W thaliana is homologous to EBP but
not to ERG2 (G. Yamamoto, un-
published results). Why do plants
and mammals need different pro-
teins for virtually identical catalytic
lumen
steps? Perhaps steric requirements
0 charged (+) necessitated this change because
0 charged (-) fungal ERG2 proteins remove the
0 hydrophobic
7a-hydrogen in the B-ring, whereas
Fig.1.Prrmary structure and transmembrane topology are common structural features of some, but not
in plants and mammals the
all, sigma receptors. a: the amino acid sequence of the guinea-pig sigma, receptor (GP) and the yeast 7B-hydrogen is lost upon EBP-
sterol C,-C, isomerase (SC) aligned as shown are 30% identical (I). and 69% similar j: and denote high mediated isomerization20.
and low similarity, respectively). The alignment was calculated using the Wisconsrn Sequence Analysis
Package according to the algorithm of Needleman and Wunsch (Genetics Computer Group, program GAP).
b,c: The proposed transmembrane topology of the sigma, receptor (red line, GenBank Accession number The sigma, receptor: also a
266537). based on the hydrophobicity plot, is identical to sterol C,& isomerases from Saccharomyces sterol C,-C, isomerization-
cerevisiae, Magmporthe grisea and W/ago maydis(blue lines, GenBank Accession numbers M74037.
222775, 217311, respectively) (b)and distinct from the mammalian sterol C,C, isomerase (blue lines,
related enzyme?
GenBank Accession numbers 237985.237986, X97755)(c). The putative transmembrane segments of the The sigma, receptor shares many
ERG2 protein (I) and the EBP (I-IV) as well as two hydrophobrc stretches common to all ERG.2proteins pharmacological (Table 1) and bio-
(a,b) are indicated. Hydrophobicity plots were calculated as described* d: The mammalian sterol CC,
isomerase (EBP) has four putative transmembrane segments EBP. emopamil binding protein
chemical (localization in the endo-
plasmic reticulum, molecular mass

6 8 TiPS - March 1997 (Vol. 18)

 CURRENT
of about 20 kDa, high density in liver
and endocrine tissues) properties with
other mammalian sigma receptors.
The major obstacle to its purification
is the lack of a selective, high-affinity
radioligand. It is tempting to specu-
late that it will be structurally related
either to EBP or the sigma, receptor.
The sigma, receptor: a
brain-specific isoform of
the isomerase?
The sterol C&, isomerase activ-
ity of two of the three cloned mem-
bers of the sigma-receptor family is
unequivocal. The structural relation-
ship between the sigma, receptor
and its yeast counterpart, as well
as their common pharmacological
properties, strongly imply a related
biochemical activity. In addition, the
high density of sigma, receptors in
the brain suggests a brain-specific
function. In contrast to the liver, the
brain depends on intracerebral de
no~osynthesis of cholesterol because
the blood-brain barrier prevents low-
density lipoprotein (LDL) receptor-
mediated cholesterol uptake from
the circulation. Cholesterol cannot
leave the brain without a unique
24S-hydroxylation stepzl. Therefore,
cerebral cholesterol synthesis can be
expected to be tightly regulated.
However, the peculiarities of brain
sterol biosynthesis are not yet under-
stood and most of the enzymes are
not yet cloned. The sigma, receptor
could represent a brain-specific
sterol C,-C, isomerase with control
mechanisms diverse from EBP (e.g.
requiring an additional regulatory
protein). This postulated subunit
could be identical to the positive
regulator of [3H](t)-pentazocine
binding, previously identified by
radiation inactivation of native
sigma, receptors. The targeted dis-
ruption of the murine gene should
definitely establish the hypothesized
sterol C,-C, isomerization-related
activity of sigma, receptors.
Sterol biosynthesis: a
pharmacological target revisited
3-Hydroxy-3-methylglutaryl-
coenzyme A (HMG-CoA)-reductase
inhibitors, such as mevastatin,
AWARENESS
provided major clues for the under-
standing of presqualene sterol
metabolism. The recent discovery
that defects in the delta7- or delta24-
reductase genes (Fig. 2) are the com-
mon cause of Smith-Lemli-Opitz
syndrome, an inborn disorder of
OH
intellectual and sexual develop-
menP, demonstrated the vital
,sterol C,-C, isomerase
importance of postsqualene choles-
+
terol biosynthesis. This stimulated
delta24-reductase
the interest in experimental drugs,
such as AY9944 and triparanol23,
which interfere with the last steps of
cholesterol biosynthesis. Cholesterol
is not only an important membrane
constituent and precursor for steroid OH Le8 7
A---
lathosterol
hormones or bile acids; it is also
involved in signal transduction. Sur- deIta5dehydrogenase
prisingly, sterol signalling is not
restricted to the nuclear transcrip- delta7-re+ductase
tion factor-mediated regulation of v
sterol biosynthesis2. Sterol regula-
tory element binding proteins are
also present in the membrane of the
endoplasmic reticulum24. The dis-
covery that dimethylzymosterol is a OH
diffusible mediator of oocyte meiosis= Fig. 2. The last four steps of postsqualene cholesterol biosynthesis
and that cholesterol is covalently in mammals. The isomerlzation reaction catalysed by the emopamll
attached to a hedgehog protein binding protein (EBP), the mammalian sterol C,-C, lsomerase, is
shown in red In fungi that synthesize ergosterol. instead of chol-
essential for brain morphogenesis2
esterol, an essentially identical isomerizatlon step is mediated by the
suggests exciting, new and vital ERG2 protein. Whereas in plants and mammals the 7@-hydrogen is
functions of sterols. lost upon EBP-mediated isomerlzation, fungal ERG2 proteins remove
the 7a-hydrogen.
Perspectives for drug
development function is sterol biosynthesis; what
If we hypothesize that the com- are the implications for drug devel-
mon denominator of sigma receptor opment? Widely used agricultural
Table 1. Pharmacological relatedness of members of the sigma receptor
family
Sigma, receptor EBP ERG2 protein
4 (nr4 It& M4 4 (nr4)
lfenprodil
Opipramol i.2 Z ;7
(4Emopamil 1 6 65
Amiodarone 1 25 62
Trifluoperazine 15 13 500
Haloperidol 0.2 190 0.5
(*I-Pentazocine 1.7 500 1000
Guinea-pig sigma, receptor, human sterol C& isomerase IEBP) and yeast sterol C,-C, lsomerase
(ERG2 protein) share a high affinity for ifenprodil. opipramol, emopamil and amiodarone. Further-
more, the sigma, receptor shares with EBP the high affinity for trlfluoperazine and with the ERG2 pro-
tein the subnamolar affinity for haloperidol. aAdditionally, the results for the sigma, receptor, K, or
ICs, [nMl, were as follows: ifenprodil. n.d.; epipramol, 6; (t)-emopamil, 9; amiodarone, n.d.; trifluop-
erazine. n.d.; haloperldol. 0.6; pentazocine, 60. EBP, emopamil binding protein; n.d, not determined
Data from Fiefs 10, 11, 13, 16.
TIPS - March 1997 Wol 18) 6 9
 CURRENT
fungicides of the morpholine class
inhibit fungal growth by interfering
with sterol C,-C, isomerization. The
morpholine analogue amorolfine is
already licensed for topical treat-
ment of onychomycosis in man.
Potent systemic antimycotics are
urgently needed as substitutes for
antifungal chemotherapeutics of the
polyene (amphotericin B) and azole
class (ketoconazole, fluconazole) as
severe side-effects, or fungal resist-
ance, limit their use. Selective
inhibitors of fungal sterol C,-C,
isomerases which do not interfere
with human sterol biosynthesis can
now be developed, employing
sigma-ligand binding assays, in
addition to recombinant fungal and
human isoenzymes.
In mammals, sterol biosynthesis
inhibitors impair cell growth pre-
sumably because sterols are
required for cell division26. More-
over, regulators of sterol biosynthe-
sis are involved in apoptotic cell
death27, which explains the cyto-
toxicity and immunosuppression
elicited by sigma ligandW9. In cer-
tain pathophysiological conditions
Acknowledgements such effects might be desirable. As
The authors research
was suppolted by a
briefly outlined, the brain depends
Baehrlnger-lngelhelm on the de nova synthesis of choles-
fellowship and grants
from the Osterretchische
terol. Sigma ligands exert anti-
Natmnalbank and ischaemic effects in animal models
Fends mrF~%derung der
Wlssenschaftllchen
of strokeao;it is conceivable that inhi-
Farschung bition of postsqualene sterol biosyn-
Four f3-adrenoceptor subtypes in the
mammalian heart
Albert0 J. Kaumann
Since its proposal in 1989, evidence
has been accumulating for the exist-
ence and function of a third p-
adrenoceptor that, besides coexist-
ing with pr- and p,-adrenoceptors,
A. J. Kaumann. mediates cardiostimulation in sev-
Senior Research eral species, including man. This
Officer.
atypical cardiac p-adrenoceptor can
The Babraham
Institute, Cambridge, couple to the G, protein/adenylate
UK CB24AT cyclase pathway, as p,- and &-
70 TiPS - March 1997 (Vol. 18)
AWARENESS
thesis can modulate neuronal or glial
proliferation and block neuronal
death programmes. Changes in the
lipid composition of cerebral mem-
branes are expected to alter neuronal
excitability. Therefore, the long-term
effects of the inhibition of post-
squalene sterol biosynthesis remain
to be investigated. It is conceivable
that such effects could be exploited
therapeutically to treat mental dis-
orders such as psychosis. This ren-
ders cholesterol biosynthesis in the
brain and the growing family of
sigma receptors fascinating targets
for future drug development.
Selected references
Quirion, R. et al. (1987) Trends Neurosci. 10,
444446
Chavkin, C. (1990) Trends Phurmncol. Sci.
11,213-215
Walker, J. M. ef aI. (1990) Pharttiacol. Rev.
42,355402
Itzhak, Y., ed. (1994) Sigma Receptors,
Academic Press
Su, T-P., London, E. D. and Jaffe, J. H.
(1988) Science 240,219-221
Schwarz, S., Pohl, I. and Zhou, G-Z. (1989)
Science 246,1635-1637
Martin, W. R., Eades, C. E., Thompson,
J.A., Huppler, R. E. and Gilbert, I. E.
(1976) J. PhurmncoL Exp. Ther. 197,517-532
Quirion, R. et RI. (1992) Trends Phurmacol.
Sci. 13,8%36
de Costa, B. R. et RI. (1989) FEBS Lett. 251,
53-58
Moebius, F. F. et al. (1993) Mol. Dhnrmucof.
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Hanner, M. et al. (1996) Proc. Nut/. Amd.
Sci. U. S. A. 93,8072-8077
Silve, S. et al. (1996) Mol. Cell. Biol. 16,
2719-2727
adrenoceptors usually do. This
contrasts with recent reports for the
existence of /3,-adrenoceptors, in
the human ventricle, that couple
to the Gi protein and mediate
cardiodepressant effects. Here, the
pharmacological properties of the
four P-adrenoceptor subtypes are
compared and their function
discussed.
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13 Moebius, F. F. et ~2. (1996) Biochemistry 51,
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14 Parks, L. W., Smith, S. J. and Crowley, J. H.
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15 Zech, C., Staudinger, R., Muhlbacher, J.
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16 Moebius, F. F., Burrows, G. G., Striessnig,
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col. 43,139-148
17 Moebius, F. F. et al. (1994) /. Biol. Chem. 269,
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18 Hanner, M. et ~1. (1995) 1. Biol. Ckmr. 270,
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19 Silve, S. et al. (1996) I. Biol. C!irm. 271,
22434-22440
20 Benveniste, P. (1986) Annu. Rezl. Phf
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21 Lutjohann, D. et al. (1996) Proc. Nat/. Acad.
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22 Clayton, I., Mills, K., Keeling, J. and
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23 Janowski, B. A., Willy, I J., Devi, T. R.,
Falck, J. R. and Mangelsdorf, D. J. (1996)
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W. D. (1995) 1. Neurosci. 15, 117-134
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Chemical names
AY99ti 1,4-bis(2-chlorobenzyl-
aminomethyl)cyclohexane
SKF10047: N-allylnormetazocine
SR31747: cis-N-cyclohexyl-N-ethyl-
3-(3-chloro-4-cyclohexylphenyl)
propen-2-ylarnine hydrochloride
Cardiac pl- and
P,-adrenoceptors
Activation of coexisting &- and
l3,-adrenoceptors can modify car-
disc function in both beneficial and
harmful ways. In the human heart,
both receptors mediate the increases
in contractile force-3 and sinoatrial
rate that occur, for example, in situ-
ations of stress, but can also mediate
arrhythmias that can be mimicked
experimentallys. In human heart tis-
sue& and myocytess, both &- and
P,-adrenoceptors also accelerate
myocardial relaxation, thereby
improving diastolic function. In the
heart of other species, however, only
PII: SO165-6147(96)01033-J