Research work of Javitch group
The long-term goals and research objectives of
my laboratory’s research are: 1) To understand the structural
bases of agonist and antagonist binding and specificity in the dopamine
D2-like receptors and related biogenic amine receptors. 2) To determine
how agonist binding is transduced into G-protein activation. 3)
To determine the structural bases of the transport of substrate
by the dopamine transporter and its inhibition by drugs such as
cocaine. In the pursuit of these objectives we are carrying out
research on several GPCRs and sodium-dependent transporters, which
is funded principally by the National Institute of Mental Health
and the National Institute on Drug Abuse, respectively.
We have mapped the entire binding-site crevice of the dopamine D2
receptor, which is formed and lined by the transmembrane segments
(TMs) (Javitch et al. 1994; Javitch et al. 1995; Javitch et al.
1995; Fu et al. 1996; Javitch et al. 1998; Javitch et al. 1999;
Shi et al. 2001; Javitch et al. 2002). Our identification of all
the residues that are water-accessible in this binding-site crevice
led to our identification of structural determinants of pharmacological
specificity in the dopamine D2 and D4 receptors (Simpson et al.
1999). In a D2 receptor background, mutations of clustered residues
in TM2, TM3, and TM7 to the aligned D4-receptor residues increased
the affinity of the mutant D2 receptor for D4-selective ligands
by three orders of magnitude.
We studied transduction in the ß2 adrenergic receptor where
we showed that conformational changes in TM6 are associated with
receptor activation and demonstrated that the presence of an “ionic
lock” between the cytoplasmic ends of TM3 and TM6 stabilizes
the inactive state of the ß2 adrenergic receptor (Ballesteros
et al. 2001). We have also demonstrated that, through specific interactions
in its different rotamer configurations, the highly conserved Cys(6.47)
in TM6 modulates the configuration of a cluster of nearby aromatic
residues and the TM6 proline-kink, forming part of a rotamer toggle
switch that modulates receptor activation (Shi et al. 2002).
The structural implications of our work on the D2 receptor and the
ß2 receptor are remarkably consistent with the recent high-resolution
structure of rhodopsin (Palczewski et al. 2000; Ballesteros et al.
2001). TM4 is an exception in that residues facing lipid in rhodopsin
are water-accessible in the D2 receptor, and our recent cross-linking
data indicate that TM4 forms part of a D2 receptor homodimer interface
(Guo et al. 2003). We are currently mapping the entire interface
and studying its role in receptor activation.
Neurotransmitter reuptake by transport proteins is a major mechanism
for terminating synaptic transmission. High affinity transport systems
have been identified for the neurotransmitters dopamine, norepinephrine,
serotonin, GABA, glutamate, and glycine. These transporters require
the presence of extracellular sodium and chloride, and operate by
coupling the movement of ions down an electrochemical gradient to
the transmembrane translocation of substrate. The dopamine transporter
is the major molecular target responsible for both the rewarding
properties and abuse potential of several psychostimulants, including
cocaine, amphetamine, and methamphetamine. Despite a great deal
of effort, however, the molecular relationships between the cocaine
binding site, the binding sites of other inhibitors of dopamine
transport, and the dopamine binding site and transport pathway remain
unclear. Binding of substrate, sodium and chloride to DAT and to
related sodium- and chloride-coupled neurotransmitter transporters
evokes a conformational change that exposes the substrate and ions
to the intracellular environment where they are released. Therefore,
a water-accessible transport pathway must be formed among the membrane-spanning
segments. This pathway should be accessible to hydrophilic reagents
applied extracellularly. Using methods related to those we have
used to study the binging-site crevice of the dopamine receptor,
we are attempting to identify the amino acid residues forming the
surface of the cocaine binding site, the dopamine binding site,
and the transport pathway in the human dopamine transporter, as
well as to identify residues in loops that are conformationally
sensitive and may contribute to forming extracellular and intracellular
gates (Ferrer and Javitch 1998; Chen et al. 2000; Reith et al. 2001;
Whitehead et al. 2001; Park et al. 2002). We are also mapping the
oligomerization interface of the human dopamine transporter and
studying the functional role of DAT oligomerization (Hastrup et
al. 2001; Norgaard-Nielsen et al. 2002). In collaboration with Dr.
Aurelio Galli at Vanderbilt University and Dr. Ulrik Gether at the
University of Copenhagen, we are also studying regulation and internalization
of DAT by substrates, inhibitors, and signal transduction pathways
(Saunders et al. 2000; Carvelli et al. 2002; Daws et al. 2002; Granas
et al. 2003).
Through sequence analysis, we have identified an entire family of
proteins in archaea and in bacteria that are homologous to DAT.
From the currently available genome sequences, more than 50 proteins
from more than 20 different organisms appear to fall into this family.
Additional family members are being identified at a rapid pace with
the sequencing of additional bacterial genomes. In collaboration
with Dr. Gary Rudnick’s group at Yale University, we recently
demonstrated that one of these, TnaT from Symbiobacterium thermophilum
is a sodium-dependent tryptophan transporter (Androutsellis-Theotokis
et al. 2003). We are exploring the properties of TnaT and a subset
of these DAT archaeal and bacterial homologs in order to assess
their suitability for direct and indirect structural studies. Our
goal is to choose a limited number of these archaeal and bacterial
transporters for use in crystallization trials as a preliminary
step towards obtaining a high-resolution structure. Moreover, we
will also pursue biochemical and biophysical methods to acquire
functional data and indirect structural information about these
transporters, as model systems for the eukaryotic neurotransmitter
A., N. R. Goldberg, K. Ueda, T. Beppu, M. L. Beckman, S. Das, J.
A. Javitch and G. Rudnick (2003). "Characterization of a functional
bacterial homologue of sodium-dependent neurotransmitter transporters."
J Biol Chem 278(15): 12703-9.
2. Ballesteros, J. A., A. D. Jensen,
G. Liapakis, S. G. Rasmussen, L. Shi, U. Gether and J. A. Javitch
(2001). "Activation of the beta 2-adrenergic receptor involves
disruption of an ionic lock between the cytoplasmic ends of transmembrane
segments 3 and 6." J Biol Chem 276(31): 29171-7.
3. Ballesteros, J. A., L. Shi and J.
A. Javitch (2001). "Structural mimicry in G protein-coupled
receptors: implications of the high-resolution structure of rhodopsin
for structure-function analysis of rhodopsin-like receptors."
Mol Pharmacol 60(1): 1-19.
4. Carvelli, L., J. A. Moron, K. M.
Kahlig, J. V. Ferrer, N. Sen, J. D. Lechleiter, L. M. Leeb-Lundberg,
G. Merrill, E. M. Lafer, L. M. Ballou, T. S. Shippenberg, J. A.
Javitch, R. Z. Lin and A. Galli (2002). "PI 3-kinase regulation
of dopamine uptake." J Neurochem 81(4): 859-69.
5. Chen, N., J. V. Ferrer, J. A. Javitch
and J. B. Justice, Jr. (2000). "Transport-dependent accessibility
of a cytoplasmic loop cysteine in the human dopamine transporter."
J Biol Chem 275(3): 1608-14.
6. Daws, L. C., P. D. Callaghan, J.
A. Moron, K. M. Kahlig, T. S. Shippenberg, J. A. Javitch and A.
Galli (2002). "Cocaine increases dopamine uptake and cell surface
expression of dopamine transporters." Biochem Biophys Res Commun
7. Ferrer, J. V. and J. A. Javitch
(1998). "Cocaine alters the accessibility of endogenous cysteines
in putative extracellular and intracellular loops of the human dopamine
transporter." Proceedings of the National Academy of Sciences
of the United States of America 95(16): 9238-9243.
8. Fu, D., J. A. Ballesteros, H. Weinstein,
J. Chen and J. A. Javitch (1996). "Residues in the seventh
membrane-spanning segment of the dopamine D2 receptor accessible
in the binding-site crevice." Biochemistry 35(35): 11278-85.
9. Granas, C., J. Ferrer, C. J. Loland,
J. A. Javitch and U. Gether (2003). "N-terminal truncation
of the dopamine transporter abolishes phorbol ester- and substance
P receptor-stimulated phosphorylation without impairing transporter
internalization." J Biol Chem 278(7): 4990-5000.
10. Guo, W., L. Shi and J. A. Javitch
(2003). "The fourth transmembrane segment forms the interface
of the dopamine D2 receptor homodimer." J Biol Chem 278(7):
11. Hastrup, H., A. Karlin and J. A.
Javitch (2001). "Symmetrical dimer of the human dopamine transporter
revealed by cross-linking Cys-306 at the extracellular end of the
sixth transmembrane segment." Proc Natl Acad Sci U S A 98(18):
12. Javitch, J. A., J. A. Ballesteros,
J. Chen, V. Chiappa and M. M. Simpson (1999). "Electrostatic
and aromatic microdomains within the binding-site crevice of the
D2 receptor: contributions of the second membrane-spanning segment."
Biochemistry 38: 7961-7968.
13. Javitch, J. A., J. A. Ballesteros,
H. Weinstein and J. Chen (1998). "A cluster of aromatic residues
in the sixth membrane-spanning segment of the dopamine D2 receptor
is accessible in the binding-site crevice." Biochemistry 37(4):
14. Javitch, J. A., D. Fu and J. Chen
(1995). "Residues in the fifth membrane-spanning segment of
the dopamine D2 receptor exposed in the binding-site crevice."
Biochemistry 34(50): 16433-9.
15. Javitch, J. A., D. Fu, J. Chen
and A. Karlin (1995). "Mapping the binding-site crevice of
the dopamine D2 receptor by the substituted-cysteine accessibility
method." Neuron 14(4): 825-31.
16. Javitch, J. A., X. Li, J. Kaback
and A. Karlin (1994). "A cysteine residue in the third membrane-spanning
segment of the human D2 dopamine receptor is exposed in the binding-site
crevice." Proceedings of the National Academy of Sciences of
the United States of America 91(22): 10355-9.
17. Javitch, J. A., L. Shi and G. Liapakis
(2002). "Use of the substituted cysteine accessibility method
to study the structure and function of G protein-coupled receptors."
Methods Enzymol 343: 137-56.
18. Norgaard-Nielsen, K., L. Norregaard,
H. Hastrup, J. A. Javitch and U. Gether (2002). "Zn(2+) site
engineering at the oligomeric interface of the dopamine transporter."
FEBS Lett 524(1-3): 87-91.
19. Palczewski, K., T. Kumasaka, T.
Hori, C. A. Behnke, H. Motoshima, B. A. Fox, I. Le Trong, D. C.
Teller, T. Okada, R. E. Stenkamp, M. Yamamoto and M. Miyano (2000).
"Crystal structure of rhodopsin: A G protein-coupled receptor."
Science 289(5480): 739-45.
20. Park, S. U., J. V. Ferrer, J. A.
Javitch and D. M. Kuhn (2002). "Peroxynitrite inactivates the
human dopamine transporter by modification of cysteine 342: potential
mechanism of neurotoxicity in dopamine neurons." J Neurosci
21. Reith, M. E., J. L. Berfield, L.
C. Wang, J. V. Ferrer and J. A. Javitch (2001). "The uptake
inhibitors cocaine and benztropine differentially alter the conformation
of the human dopamine transporter." J Biol Chem 276(31): 29012-8.
22. Saunders, C., J. V. Ferrer, L.
Shi, J. Chen, G. Merrill, M. E. Lamb, L. M. Leeb-Lundberg, L. Carvelli,
J. A. Javitch and A. Galli (2000). "Amphetamine-induced loss
of human dopamine transporter activity: an internalization-dependent
and cocaine-sensitive mechanism." Proc Natl Acad Sci U S A
23. Shi, L., G. Liapakis, R. Xu, F.
Guarnieri, J. A. Ballesteros and J. A. Javitch (2002). "beta
2 Adrenergic receptor activation: Modulation of the proline kink
in TM6 by a rotamer toggle switch." J Biol Chem.
24. Shi, L., M. M. Simpson, J. A. Ballesteros
and J. A. Javitch (2001). "The first transmembrane segment
of the dopamine D2 receptor: accessibility in the binding-site crevice
and position in the transmembrane bundle." Biochemistry 40(41):
25. Simpson, M. M., J. A. Ballesteros,
V. Chiappa, J. Chen, M. Suehiro, D. S. Hartman, T. Godel, L. A.
Snyder, T. P. Sakmar and J. A. Javitch (1999). "Dopamine D4/D2
receptor selectivity is determined by a divergent aromatic microdomain
contained within the second, third, and seventh membrane-spanning
segments." Mol Pharmacol 56(6): 1116-26.
26. Whitehead, R. E., J. V. Ferrer,
J. A. Javitch and J. B. Justice (2001). "Reaction of oxidized
dopamine with endogenous cysteine residues in the human dopamine
transporter." J Neurochem 76(4): 1242-51.