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 transporters.

1. Androutsellis-Theotokis, 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 290(5): 1545-50.
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): 4385-8.
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): 10055-60.
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): 998-1006.
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 22(11): 4399-405.
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 97(12): 6850-5.
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): 12339-48.
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.