Download Erythrocyte G Protein–Coupled Receptor Signaling in Malarial

Erythrocyte G Protein–Coupled
Receptor Signaling in
Malarial Infection
Travis Harrison,1,2 Benjamin U. Samuel,1,2*
Thomas Akompong,1,2 Heidi Hamm,3 Narla Mohandas,4
Jon W. Lomasney,1† Kasturi Haldar1,2†
Erythrocytic mechanisms involved in malarial infection are poorly understood. We
have found that signaling via the erythrocyte ␤2-adrenergic receptor and heterotrimeric guanine nucleotide– binding protein (G␣s) regulated the entry of the
human malaria parasite Plasmodium falciparum. Agonists that stimulate cyclic
adenosine 3⬘,5⬘-monophosphate production led to an increase in malarial infection
that could be blocked by specific receptor antagonists. Moreover, peptides designed
to inhibit G␣s protein function reduced parasitemia in P. falciparum cultures in
vitro, and ␤-antagonists reduced parasitemia of P. berghei infections in an in vivo
mouse model. Thus, signaling via the erythrocyte ␤2-adrenergic receptor and G␣s
may regulate malarial infection across parasite species.
Plasmodium falciparum is a protozoan parasite that causes the most virulent form of
human malaria. It infects both hepatocytes
and mature red blood cells, but the erythrocytic stages of infection are responsible for
all of the symptoms and pathologies associated with the disease (1). Parasite invasion is
a complex, multistep process where the host
erythrocyte membrane undergoes involution
and deformation, followed by invagination
and swelling (2). However, uninfected erythrocytes are incapable of pinocytocis or endocytosis, and host pathways are not known to be
involved in signaling the entry of P. falciparum.
Heterotrimeric guanine nucleotide– binding
regulatory proteins (G proteins) constitute a
well-characterized class of signal transduction
proteins in mammalian cells (3). They regulate
important cellular processes ranging from transcription, motility, secretion, and contractility
(4). G proteins reside at the cytoplasmic face of
the cellular plasma membrane, where they can
couple with a variety of transmembrane receptors to transduce extracellular signals initiated
by many hormones, neurotransmitters, chemokines, and autocrine and paracrine factors to a
wide range of effectors within the cell (5). G
proteins are activated by guanine nucleotide
exchange factors (GEFs), which promote the
dissociation of guanosine diphosphate (GDP)
Department of Pathology, 2Department of Microbiology-Immunology, Feinberg School of Medicine,
Northwestern University, 303 Chicago Avenue, Chicago, IL 60611, USA. 3Department of Pharmacology,
Vanderbilt University, Nashville, TN 37240, USA.
4
New York Blood Center, New York, NY 10021, USA.
1
*Present address: Department of Ophthalmology and
Visual Sciences, University of Chicago, Room 103
Visual Science Center, Chicago, IL 60637, USA.
†To whom correspondence should be addressed. Email: [email protected] (K.H.); j-lomasney@
northwestern.edu ( J.W.L.)
1734
from the inactive G protein and replacement
with guanosine triphosphate (GTP). G protein–
coupled receptors (GPCRs) act as GEFs for
large heterotrimeric G proteins. Four major
families of ␣ subunits—G␣s, G␣i/o, G␣q/11,
and G12/13— have been described and each
specifies a distinct set of downstream signals
(4). Although G proteins have been intensively
studied in a wide range of cells, their functions
in mature red blood cells are poorly understood.
These cells are enucleated, have no intracellular
structures, and are incapable of de novo protein
and lipid biosynthesis (6, 7).
The G␣s present in erythrocytes can be
isolated in detergent-resistant membrane
(DRM) rafts and is recruited to the malarial
vacuole (8). Another heterotrimeric protein,
G␣q, is also present in the red blood cell but
does not concentrate at the malarial vacuole
(Fig. 1). The G␣s-coupled ␤2-adrenergic receptor (␤2-AR), which is detected in DRMs (9),
was also recruited to the vacuolar parasite (Fig.
1). To determine whether the recruitment of
G␣s to the plasmodial vacuole may have functional consequences for infection, we introduced into cocultures of parasites and erythrocytes peptides derived from the C-terminal
region of G␣s that block the interaction of this
G protein with its activating receptors. The last
11 amino acids of the G protein are critical for
interaction with GPCR (10) and competitively
block G␣ association with GPCRs and abrogate
downstream signaling events. G␣s peptide
[QRMHLRQYELL (11)] reduced infection of
erythrocytes by P. falciparum by 87% (Table
1). In contrast, the scrambled G␣s (G␣scr),
which contained the same amino acids as the
G␣s peptide but in a different sequence
[ELRLQHYMQLR (11)], inhibited infection
by less than 5% (Table 1). Thus, the inhibition
of infection effected by the G␣s peptide was
dependent on its sequence, and the peptide
probably blocked G␣s function (12). Database
searches failed to reveal G␣s (or other heterotrimeric G proteins, although Rab GTPases are
present) in the P. falciparum genome (13). One
report suggests the presence of plasmodial heterotrimeric G proteins (14), but there are no
parasite heterotrimeric G proteins that are homologous to mammalian G proteins, and the
G␣s peptides used here were expected to selectively disrupt host G␣s protein function.
Erythrocytic infection by P. falciparum
is initiated when the extracellular merozoite stage enters the red blood cell to form an
intracellular ring. The G␣s peptide displayed a dose-dependent inhibition of new
ring formation (table S1). Intracellular development of rings through “trophozoite” and terminal “schizonts” stages (that rupture to release
merozoites) remained unaffected, suggesting
that the addition of the G␣s peptide blocked a
step in erythrocyte entry. To determine how
peptides gained access to the erythrocyte, we
preincubated fluorescent [fluorescein isothiocyanate (FITC)–labeled] forms of G␣s and
G␣scr peptides (fig. S1) with red blood cells or
late-stage schizonts and segmenters for four
hours and subsequently used them in an infection assay. This pretreatment failed to block
infection (fig. S2), suggesting that the peptides,
though acetylated, could not enter cells directly.
However, when FITC-labeled peptides were
added to the invasion assay, fluorescence was
found in red blood cells associated with parasites (Fig. 2A). Staining with antibodies to
MSP1 (a protein on the surface of invasive
merozoites) detected extracellular parasites
blocked in entry. Although this assay underestimates the block in parasite entry (because
extracellular merozoites are prone to degradation) in incubations containing FITC-G␣s, 75%
of the parasites were detected by the antibodies
to MSP1 (Fig. 2A, a). In incubations containing
Fig. 1. Distribution of endogenous G␣s, G␣q,
and ␤2-AR (green) in newly formed P. falciparum-ring-infected erythrocytes as detected by
indirect immunofluorescence assays (8). Parasite (P) nucleus is Hoechst-stained (blue); arrowhead indicates red-cell plasma membrane.
Scale bar indicates 3 ␮m.
19 SEPTEMBER 2003 VOL 301 SCIENCE www.sciencemag.org
Downloaded from www.sciencemag.org on May 7, 2015
REPORTS
REPORTS
FITC-G␣scr, 90% of the parasites were not
detected by the antibodies to MSP1, indicating
that they were intracellular (Fig. 2A, b). Thus,
peptides gained access to red blood cells at the
time of parasite entry (Fig. 2B), probably across
the nascent vacuole; the vacuolar membrane has
been shown to have altered permeability (15).
Peptides that block interaction of G␣s with
its receptors also block malarial infection. Thus,
activation of G␣s via its receptors may influence malarial infection. The two major G␣sassociated receptors known to be present on red
blood cells are the ␤-ARs and the adenosine
receptors. Agonists of both the ␤2-ARs and
adenosine receptors stimulated infection of P.
falciparum (3D7 strain) in vitro about twofold
(Fig. 3A). Stimulation was dose-dependent (fig.
S3). Competitive antagonists blocked this stimulation. A combination of a ␤2-AR agonist and
adenosine receptor agonist showed additive effects in stimulating infection. G␣s peptides
blocked infection by 80 to 90%, suggesting that
the receptors were mediating their effects via
G␣s. The same level of agonist-stimulated infection, and its inhibition by antagonists, was
detected when another strain of P. falciparum
(FCB) was used (Fig. 3B), suggesting that the
mechanisms of G protein regulation of infection have been conserved across independent
strains of P. falciparum.
To confirm that G␣s-coupled receptors signal in red blood cells, we demonstrated that
agonists for both the ␤2-ARs and adenosine
receptors stimulated cyclic adenosine monophosphate (cAMP) accumulation in red blood
cells (Fig. 3C). Thus, both GPCRs were functional in erythrocytes, and inhibition of host
G␣s signaling in erythrocytes blocked malarial
infection. To investigate further the pharmacology of receptor-mediated inhibition of infection, we tested the effects of inactive stereoisomers, neutral antagonists, and inverse agonists
of ␤2-AR. An inverse agonist is a compound
with a negative intrinsic activity, i.e., a compound that produces conformational changes in
the receptor that are less favorable to activation
of G proteins than the ground state (16). Although racemic preparations for the antagonist
propranolol were active in blocking (agonist)
isoproterenol-stimulated infection, there was no
Table 1. Effects of C-terminal peptides of human
heterotrimeric G␣s on P. falciparum infection of
erythrocytes. In vitro infection assays were incubated with the indicated peptides (G␣s or G␣scr)
or mock treated (25). Infection is shown relative to
that seen with mock cultures; parasitemia in mock
cultures was at ⬃20%. Standard error is 10%.
Cultures incubated with G␣s peptide were inhibited in new ring formation.
Peptide
G␣s
G␣scr
Sequence
Inhibition of
infection
QRMHLRQYELL (11)
ELRLQHYMQLR (11)
87%
4%
effect with the (⫹) inactive stereoisomer of
propranol (Fig. 3D). Thus, inhibition of infection was because of propranolol acting at the
receptor and not any nonspecific membrane
effects. The neutral antagonist alprenolol was
slightly less active than racemic (⫹/–) propranolol, whereas the inverse agonist ICI 118,551
(ICI) was more efficacious and could even reduce baseline levels of infection by about 50%.
The greater efficacy of ICI suggests the existence of precoupling of receptors to G proteins.
Although theoretically such inverse agonists
should be more efficacious, this has yet to be
shown in many systems (17, 18).
The in vitro studies led us to investigate
whether G␣s receptor antagonists could influence parasite proliferation in a mouse model
using P. berghei, a rodent malaria parasite (19).
For racemic propranolol, the median inhibitory
concentration (IC50) appeared to be a dose of
7.5 mg/kg (administered twice daily; fig. S4A).
The median lethal dose (LD50) of propranolol
for intravenous injection into rodents is 470
mg/kg per day, and the LD50 for intraperitoneal
injection (the route used here) was expected to
be even higher, suggesting that these compounds were well tolerated. At 7.5 m/kg, the
inactive (⫹) propranolol isomer had no effect
on in vivo infection; the neutral antagonist alprenol showed a reduction of ⬃30%, whereas
inverse agonist ICI inhibited parasitemia by
⬃50% (fig. S4B). This trend of inhibition was
consistent with the inhibitory effects of these
compounds seen on agonist-stimulated in vitro
Fig. 2. Model for G peptide translocation into erythrocytes during parasite
infection. (A) FITC-G␣s (a) or FITCG␣scr (G␣s-scrambled) (b) peptides
(green) were added to an in vitro infection assay, probed with antibodies to
MSP1 (red) to detect extracellular parasites, and scored (25). Parasite (P) nucleus is Hoechst-stained (blue). Scale
bar, 3 ␮m. (B) A schematic drawing of
G␣s inhibition of ring formation. On the
basis of data in Fig. 2A, table S2, fig. S1,
and fig. S2, we propose that the peptide
was taken in with the parasite and
translocated across the nascent or newly formed vacuole. Presence of the
FITC-G␣s peptide prevented intracellular ring formation, whereas FITC-G␣scr allows intracellular ring formation.
Fig. 3. Effects of agonists and antagonists
of the ␤-ARs and adenosine receptors on
infection of P. falciparum and cAMP production in erythrocytes. (A) P. falciparum
(strain 3D7) infection of erythrocytes in
cultures that are mock-treated control (C)
cultures or treated with agonists and antagonists of the ␤2-AR [isoproterenol (I)agonist and racemic (⫹/–) propranolol (P)antagonist] or agonists and antagonists of
the adenosine receptor [5⬘-N-ethylcarboxamidoadenosine (N) and adenosine (A) are
agonists; 8-( p-sulfophenyl)theophilline (S) is an antagonist] (25). With the exception of adenosine
(which was used at 1 mM), effective concentrations used for agonists and antagonists were at 10⫺5
M (suggesting a low abundance of receptors on erythrocytes). Changes are shown relative to
control cultures, which achieved parasitemias of 9 to 11% or greater (25). Standard error is 10%
in data from triplicate assays. (B) P. falciparum (strain FCB) infection of erythrocytes in cultures
under conditions described in (A). (C) cAMP production upon activation of ␤2-ARs and adenosine
receptors in infected erythrocytes was measured with the use of the Direct cAMP Enzyme
Immunoassay (Assay Designs, Incorporated, Ann Arbor, MI) kit (25). A two- to fourfold stimulation
in cAMP production was seen in triplicate assays. (D) Specificity of various ␤2-AR antagonists for
P. falciparum infection. Isoproterenol (I) and racemic (⫹/–) propranolol (P) were tested, along with
inactive propranolol isomer (⫹P), neutral antagonist alprenol (Al), and inverse agonist ICI (⫾)-1[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol (Ic). All agonists
and antagonists were used at 10⫺5 M. Infection assays were as described in (A).
www.sciencemag.org SCIENCE VOL 301 19 SEPTEMBER 2003
1735
REPORTS
infections of P. falciparum. Thus, signaling via
erythrocyte ␤2-AR and G␣s activation appears
to be conserved across parasite species.
Although we found some degree of precoupling of ␤2-AR to G␣s in erythrocytes (evidenced by efficacy of neutral antagonists in
vitro), there appeared to be stimulation of receptors during infection in vivo. How erythrocyte G␣s-coupled receptors are stimulated during malarial infection in vivo remains to be
understood. One possibility is that catecholamine levels are augmented during infection,
resulting in stimulation of receptors such as
␤2-AR. An increase of catecholamines may
come from an elevation of the sympathetic
response or the production of catecholaminelike molecules by the malaria parasite. This
may explain why antagonists like propranolol
are effective at reducing parasitemias in vivo.
The use of erythrocyte G-protein raftassociated signaling mechanisms in malarial
entry and/or establishment of the vacuole may
provide a reason why (i) erythrocyte rafts are
required and (ii) their resident proteins are internalized in infection (8, 9, 20). Raft-dependent G-protein signaling has been demonstrated
in cells (21). Although the physiological functions of G protein receptors and their associated
signaling mechanisms in erythrocytes are not
well understood, an emerging idea is that they
may contribute to interactions with endothelial
cells (22). Because both G␣s and ␤2-AR were
internalized and associated with the vacuolar
parasite, their activation in malarial infection
may regulate a step of vacuole formation that is
conserved across parasite species. This may
explain why the same antagonists inhibit infection by human malarias like P. falciparum and
rodent malaria parasites like P. berghei. Signaling via G proteins rapidly reorganizes the cellular cytoskeleton in nucleated mammalian
cells (23, 24). In P. falciparum–infected erythrocytes, nearly all skeletal components and attached integral proteins associated with the host
plasma membrane are excluded from the vacuole. Further parasite entry culminating in intravacuolar residence occurs within minutes.
Thus, signaling via GPCR may underlie the
rapid and dynamic reorganization of submembranous cytoskeleton required for infection of
the nonendocytic, mature erythrocyte by this
major human pathogen.
References and Notes
1. L. H. Miller, D. I. Baruch, K. Marsh, O. K. Doumbo,
Nature 415, 673 (2002).
2. J. A. Dvorak, L. H. Miller, W. C. Whitehouse, T. Shiroshi, Science 187, 748 (1975).
3. H. E. Hamm, J. Biol. Chem. 273, 669 (1998).
4. S. R. Neves, P. T. Ram, R. Iyengar, Science 296, 1636
(2002).
5. K. L. Pierce, R. T. Premont, R. J. Lefkowitz, Nature Rev.
Mol. Cell Biol. 3, 639 (2002).
6. S. L. Schrier, Clin. Haematol. 14, 1 (1985).
7. J. A. Chasis, M. Prenant, A. Leung, N. Mohandas, Blood
74, 1112 (1989).
8. S. A. Lauer et al., EMBO J. 19, 3556 (2000).
1736
9. B. U. Samuel et al., J. Biol. Chem. 276, 29319
(2001).
10. A. Gilchrist et al., J. Biol. Chem. 273, 14912 (1998).
11. Single-letter abbreviations for the amino acid residues are as follows: E, Glu; H, His; L, Leu; M, Met; Q,
Gln; R, Arg; and Y, Tyr.
12. A. Gilchrist, M. Bunemann, A. Li, M. M. Hosey, H. E.
Hamm, J. Biol. Chem. 274, 6610 (1999).
13. M. J. Gardner et al., Nature 419, 498 (2002).
14. M. Dyer, K. Day, Mol. Biochem. Parasitol. 110, 437
(2000).
15. S. A. Desai, R. L. Rosenberg, Proc. Natl. Acad. Sci.
U.S.A. 94, 2045 (1997).
16. T. Kenakin, Annu. Rev. Pharmacol. Toxicol. 42, 349
(2002).
17. R. A. Bond et al., Nature 374, 272 (1995).
18. C. Maack et al., Br. J. Pharmacol. 132, 1817 (2001).
19. W. Peters, B. L. Robinson, Ann. Trop. Med. Parasitol.
78, 561 (1984).
20. E. Nagao, K. B. Seydel, J. A. Dvorak, Exp. Parasitol.
102, 57 (2002).
21. J. M. Green et al., J. Cell. Biol. 146, 673 (1999).
22. J. E. Brittain, K. J. Mlinar, C. S. Anderson, E. P. Orringer,
L. V. Parise, J. Clin. Investig. 107, 1555 (2001).
23. S. Etienne-Manneville, A. Hall, Nature 240, 629
(2002).
24. J. F. Vanhauwe et al., J. Biol. Chem. 277, 34143
(2002).
25. Materials and methods are available as supporting
material on Science Online.
26. Supported by grants from the NIH to K.H. (AI39071
and HL69630), J.W.L. (HL55591 and HL03961), N.M.
(DK32094), and H.H. (EY06062 and EY10291).
Supporting Online Material
www.sciencemag.org/cgi/content/full/301/5640/1734/
DC1
Materials and Methods
Figs. S1 to S4
Table S1
17 July 2003; accepted 11 August 2003
Cerebellar LTD and
Learning-Dependent Timing of
Conditioned Eyelid Responses
S. K. E. Koekkoek, H. C. Hulscher, B. R. Dortland, R. A. Hensbroek,
Y. Elgersma, T. J. H. Ruigrok, C. I. De Zeeuw*
Mammals can be trained to make a conditioned movement at a precise time,
which is correlated to the interval between the conditioned stimulus and
unconditioned stimulus during the learning. This learning-dependent timing
has been shown to depend on an intact cerebellar cortex, but which cellular
process is responsible for this form of learning remains to be demonstrated.
Here, we show that protein kinase C– dependent long-term depression in
Purkinje cells is necessary for learning-dependent timing of Pavlovianconditioned eyeblink responses.
Precise timing of movements is crucial for
survival, and the central nervous system
continuously tries to optimize this timing.
Timing of movements can be learned, for
example, by conditioning an eyelid response to a conditioned stimulus (CS), such
as a tone, which continues until an unconditioned stimulus (US), such as an electrical shock or a corneal air puff, ceases. In
this paradigm, the timing of the eyelid response is ultimately determined by the interstimulus interval (ISI) between the onset
of the CS and the onset of the US. The
exact timing of conditioned responses depends on plasticity in the cerebellar cortex
(1–3). Yet, it is not clear which cellular
processes are responsible for the timing
properties of conditioned responses. Several mutant mice have been bred in which
induction of long-term depression (LTD)
(4 ) at the parallel fiber–Purkinje cell synapse is impaired, but so far it has not been
possible to investigate whether this form of
Department of Neuroscience, Erasmus MC, 3000 DR
Rotterdam, Netherlands.
*To whom correspondence should be addressed. Email: [email protected]
LTD contributes to learning-dependent timing (5–7 ). The deficit in these mutants
either was not cell-specific or was contaminated by aberrations in motor performance, and/or the temporospatial resolution of the eyelid recording method was
insufficient to detect timing differences in
mice. Here, we used transgenic mice in
which parallel fiber LTD is impaired in
Purkinje cells in vitro and in vivo by the
inhibition of protein kinase C (PKC) and in
which no motor performance or excitability
deficits have been detected (L7-PKCi mutants) (8–10). We subjected wild-type mice
and the transgenic mice to a novel method
of eyelid recording, the magnetic distance
measurement technique (11). This method
allows us to determine accurately the position of the eyelid of a mouse over time by
generating a local magnetic field that
moves with the eyelid and that is picked up
by an aligned field–sensitive chip while the
animal is freely moving.
Adult L7-PKCi mutants (C57/Bl6 background; n ⫽ 24) and wild-type littermates
(n ⫽ 24) were anesthetized with an oxygenated mixture of nitrous oxide and halothane, and a premade connector (SamTec;
19 SEPTEMBER 2003 VOL 301 SCIENCE www.sciencemag.org
Erythrocyte G Protein-Coupled Receptor Signaling in Malarial
Infection
Travis Harrison et al.
Science 301, 1734 (2003);
DOI: 10.1126/science.1089324
This copy is for your personal, non-commercial use only.
If you wish to distribute this article to others, you can order high-quality copies for your
colleagues, clients, or customers by clicking here.
Permission to republish or repurpose articles or portions of articles can be obtained by
following the guidelines here.
Updated information and services, including high-resolution figures, can be found in the online
version of this article at:
http://www.sciencemag.org/content/301/5640/1734.full.html
Supporting Online Material can be found at:
http://www.sciencemag.org/content/suppl/2003/09/17/301.5640.1734.DC1.html
A list of selected additional articles on the Science Web sites related to this article can be
found at:
http://www.sciencemag.org/content/301/5640/1734.full.html#related
This article cites 23 articles, 11 of which can be accessed free:
http://www.sciencemag.org/content/301/5640/1734.full.html#ref-list-1
This article has been cited by 45 article(s) on the ISI Web of Science
This article has been cited by 13 articles hosted by HighWire Press; see:
http://www.sciencemag.org/content/301/5640/1734.full.html#related-urls
This article appears in the following subject collections:
Microbiology
http://www.sciencemag.org/cgi/collection/microbio
Science (print ISSN 0036-8075; online ISSN 1095-9203) is published weekly, except the last week in December, by the
American Association for the Advancement of Science, 1200 New York Avenue NW, Washington, DC 20005. Copyright
2003 by the American Association for the Advancement of Science; all rights reserved. The title Science is a
registered trademark of AAAS.
Downloaded from www.sciencemag.org on May 7, 2015
The following resources related to this article are available online at
www.sciencemag.org (this information is current as of May 7, 2015 ):