Species- and Brain Region-Specific Dopamine Transporters: Immunological and Glycosylation Characteristics

Abstract: Dopamine transporters (DATs) from the caudate nucleus of four species (rat, mouse, dog, and human) and four regions of rat brain (striatum, nucleus accumbens, prefrontal cortex, and midbrain) were photoaffinity labeled and analyzed by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis for cross-reactivity to four epitope-specific rat antipeptide antibodies. Each of these antibodies varied in its efficiency at recognizing DAT. The DATs from the rat brain regions exhibited the same degree of recognition by each of the four sera, a result compatible with these proteins being the product of a single gene. The DATs from the different species were recognized by all four sera but with different efficiencies, possibly relating to amino acid sequence differences within the immunizing epitope. All of the photolabeled, immunoprecipitated DATs migrated with a molecular mass of ∼80 kDa, and no lower molecular mass forms were found. The DATs from all species and brain regions tested were shown by enzymatic deglycosylation to contain N-linked carbohydrates and sialic acids in amounts comparable with rat striatal DATs. The finding that no photolabeled DAT forms <80 kDa were isolated from membranes indicates that partially or incompletely glycosylated forms are not present, even in the midbrain cell bodies where immature forms might be expected to be found. These findings verify the utility of these anti-rat antibodies as biochemical tools for studying DATs from other species and extend our knowledge of biochemical characteristics of DATs from these species and brain regions.     

Palmitoylation controls dopamine transporter kinetics, degradation, and protein kinase C-dependent regulation

Palmitoylation is a lipid modification that confers diverse functions to target proteins and is a contributing factor for many neuronal diseases. In this study, we demonstrate using [(3)H]palmitic acid labeling and acyl-biotinyl exchange that native and expressed dopamine transporters (DATs) are palmitoylated, and using the palmitoyl acyltransferase inhibitor 2-bromopalmitate (2BP), we identify several associated functions. Treatment of rat striatal synaptosomes with 2BP using lower doses or shorter times caused robust inhibition of transport V(max) that occurred with no losses of DAT protein or changes in DAT surface levels, indicating that acute loss of palmitoylation leads to reduction of transport kinetics. Treatment of synaptosomes or cells with 2BP using higher doses or longer times resulted in DAT protein losses and production of transporter fragments, implicating palmitoylation in regulation of transporter degradation. Site-directed mutagenesis indicated that palmitoylation of rat DAT occurs at Cys-580 at the intracellular end of transmembrane domain 12 and at one or more additional unidentified site(s). Cys-580 mutation also led to production of transporter degradation fragments and to increased phorbol ester-induced down-regulation, further supporting palmitoylation in opposing DAT turnover and in opposing protein kinase C-mediated regulation. These results identify S-palmitoylation as a major regulator of DAT properties that could significantly impact acute and long term dopamine transport capacity.     

Phosphorylation of Dopamine Transporter Serine 7 Modulates Cocaine Analog Binding

As an approach to elucidating dopamine transporter (DAT) phosphorylation characteristics, we examined in vitro phosphorylation of a recombinant rat DAT N-terminal peptide (NDAT) using purified protein kinases. We found that NDAT becomes phosphorylated at single distinct sites by protein kinase A (Ser-7) and calcium-calmodulin-dependent protein kinase II (Ser-13) and at multiple sites (Ser-4, Ser-7, and Ser-13) by protein kinase C (PKC), implicating these residues as potential sites of DAT phosphorylation by these kinases. Mapping of rat striatal DAT phosphopeptides by two-dimensional thin layer chromatography revealed basal and PKC-stimulated phosphorylation of the same peptide fragments and comigration of PKC-stimulated phosphopeptide fragments with NDAT Ser-7 phosphopeptide markers. We further confirmed by site-directed mutagenesis and mass spectrometry that Ser-7 is a site for PKC-stimulated phosphorylation in heterologously expressed rat and human DATs. Mutation of Ser-7 and nearby residues strongly reduced the affinity of rat DAT for the cocaine analog (−)-2β-carbomethoxy-3β-(4-fluorophenyl) tropane (CFT), whereas in rat striatal tissue, conditions that promote DAT phosphorylation caused increased CFT affinity. Ser-7 mutation also affected zinc modulation of CFT binding, with Ala and Asp substitutions inducing opposing effects. These results identify Ser-7 as a major site for basal and PKC-stimulated phosphorylation of native and expressed DAT and suggest that Ser-7 phosphorylation modulates transporter conformational equilibria, shifting the transporter between high and low affinity cocaine binding states.     

Effect of N-linked glycosylation on hepatic lipase activity

Hepatic lipase (HL) is a secretory protein synthesized in hepatocytes and bound to liver endothelium. Previous studies have suggested that HL N-linked glycans are required for catalytic activity. To directly test this hypothesis, Xenopus laevis oocytes were used to express native rat HL or HL lacking one or both N-linked glycosylation sites. The expressed and secreted native HL had an apparent molecular mass of 53 kDa, consistent with purified rat liver HL. The mutant lacking both glycosylation sites, while poorly secreted, had an apparent molecular mass of 48 kDa, the same size observed for HL after enzymatic removal of N-linked oligosaccharides. Mutants lacking one of the two sites were intermediate in size and showed reduced secretion. Each of these expressed and secreted proteins had full catalytic activity that was inhibited by antisera to rat HL. Thus, N-linked glycosylation of rat HL, while important to lipase secretion, is not essential for the expression of lipase activity.     

Common human 5' dopamine transporter (SLC6A3) haplotypes yield varying expression levels in vivo

1. Individuals display significant differences in their levels of expression of the dopamine transporter (DAT; SLC6A3). These differences in DAT are strong candidates to contribute to individual differences in motor, mnemonic and reward functions. To identify "cis"-acting genetic mechanisms for these individual differences, we have sought variants in 5' aspects of the human DAT gene and identified the haplotypes that these variants define. 2. We report (i) significant relationships between 5' DAT haplotypes and human individual differences in ventral striatal DAT expression assessed in vivo using [(11)C] cocaine PET and (ii) apparent confirmation of these results in studies of DAT expression in postmortem striatum using [(3)H] carboxyflurotropane binding. 3. These observations support the idea that cis-acting variation in 5' aspects of the human DAT/SLC6A3 locus contributes to individual differences in levels of DAT expression in vivo. 5' DAT variation is thus a good candidate to contribute to individual differences in a number of human phenotypes.     

The role of N-glycosylation in function and surface trafficking of the human dopamine transporter

The present study addressed the role of N-linked glycosylation of the human dopamine transporter (DAT) in its function with the help of mutants, in which canonical N-glycosylation sites have been removed (N181Q, N181Q,N188Q, and N181Q,N188Q,N205Q), expressed in human embryonic kidney-293 cells. Removal of canonical sites produced lower molecular weight species as did enzymatic deglycosylation or blockade of glycosylation, and all three canonical sites were found to carry sugars. Prevention of N-glycosylation reduced both surface and intracellular DAT. Although partially or non-glycosylated DAT was somewhat less represented at the surface, no evidence was found for preferential exclusion of such material from the plasma membrane, indicating that glycosylation is not essential for DAT expression. Non-glycosylated DAT was less stable at the surface as revealed by apparently enhanced endocytosis, consonant with weaker DAT immunofluorescence at the cell surface and stronger presence in cytosol in confocal analysis of the double and triple mutant. Non-glycosylated DAT did not transport dopamine as efficiently as wild-type DAT as judged from the sharp reduction in uptake V(max), and prevention of N-glycosylation enhanced the potency of cocaine-like drugs in inhibiting dopamine uptake into intact cells without changing their affinity for DAT when measured in membrane preparations prepared from these cells. Thus, non-glycosylated DAT at the cell surface displays appreciably reduced catalytic activity and altered inhibitor sensitivity compared with wild type.     

Evaluation of human N-linked glycosylation sites in murine granulocyte-macrophage colony-stimulating factor.

Nonglycosylated murine and human granulocyte-macrophage colony-stimulating factor have a molecular mass of approximately 14.5 kDa predicted from the primary amino acid sequence. The expression of both proteins in COS cells leads to a heterogeneous population of molecules that differ in the degree of glycosylation. Both human and murine molecules contain two N-linked glycosylation sites that are situated in nonhomologous locations along the linear sequence. Despite this difference both proteins show a similar size distribution among the glycosylation variants. These studies analyze the effects of introducing in the murine protein novel N-linked glycosylation sites corresponding to those sites found in the human molecule. A panel of molecules composed of various combinations of human N-linked glycosylation sites in either the presence or the absence of murine N-linked glycosylation was compared. Substitution of a proper human N-linked glycosylation consensus sequence at Asn 24 did not result in N-linked glycosylation, nor was there any considerable effect on bioactivity. Replacement of the N-linked glycosylation consensus sequence at Asn 34 results in glycosylation similar to that found in the human molecule and causes a significant decrease in bioactivity. These data suggest that the position of N-linked glycosylation is critical for maximal bioactivity in a particular species and that the changes in position of these sites in different species probably occurred during evolution in response to changes in their receptors.     

Active human erythropoietin expressed in insect cells using a baculovirus vector: a role for N-linked oligosaccharide

Biologically active recombinant human erythropoietin has been expressed at high levels in an insect cell background. Expression involved the preparation of a human erythropoietin cDNA, the transfer of this cDNA to the Autographa californica nuclear polyhedrosis virus (AcNPV) genome under the polyhedrin gene promoter, and the subsequent infection of Spodoptera frugiperda cells with recombinant AcNPV. Erythropoietin cDNA was prepared through the expression of the human erythropoietin gene in COS cells using pSV2 and the construction of a COS cell cDNA library in bacteriophage Lambda GT10. Prior to transfer to the AcNPV genome, erythropoietin cDNA isolated from this library was modified at the 3'-terminus in order to replace genomic erythropoietin for SV40 cDNA derived from pSV2. Transfer of this cDNA to AcNPV and the infection of S. frugiperda cells with cloned recombinant virus led to the secretion of erythropoietin: based on bioassay, rates of hormone secretion (over 40 U/ml per h) were 50-fold greater than observed for COS cells. The purified recombinant product possessed full biological activity (at least 200,000 U/mg), but was of lower Mr (23,000) than human erythropoietin produced in COS cells (30,000) or purified from urine (30,000 to 38,000). This difference was attributed to the glycosylation of erythropoietin in S. frugiperda cells with oligosaccharides of only limited size. Further removal of N-linked oligosaccharides from this Mr 23,000 hormone using N-Glycanase yielded an apo-erythropoietin (Mr 18,000) which possessed substantially reduced biological activity. These results indicate that glycosylation, but not the normal processing of oligosaccharides to complex types, is required for the full hormonal activity of human erythropoietin during red cell development.     

Role of N-linked carbohydrate processing and calnexin in human hepatic lipase secretion.

The addition and endoplasmic reticulum (ER) glucosidase processing of N-linked glycans is essential for the secretion of rat hepatic lipase (HL). Human HL is distinct from rat HL by the presence of four as opposed to two N-linked carbohydrate side chains. We examined the role of N-linked glycosylation and calnexin interaction in human HL secretion from Chinese hamster ovary (CHO) cells stably expressing a human HL cDNA. Steady-state and pulse-chase labeling experiments established that human HL was synthesized as an ER-associated precursor containing high mannose N-linked glycans. Secreted HL had a molecular mass of approximately 65 kDa and contained mature N-linked sugars. Inhibition of N-linked glycosylation with tunicamycin (TM) prevented secretion of HL enzyme activity and protein mass. In contrast, incubation of cells with the ER glucosidase inhibitor, castanospermine (CST), decreased human HL protein secretion by 60%, but allowed 40% of fully active HL to be secreted. HL protein mass and enzyme activity were also recovered from the media of a CHO-derivative cell line genetically deficient in ER glucosidase I activity (Lec23) that was transiently transfected with a human HL cDNA. Co-immunoprecipitation experiments demonstrated that newly synthesized human HL bound to the lectin-like ER chaperone, calnexin, and that this interaction was inhibited by TM and CST. These results suggest that under normal conditions calnexin may increase the efficiency of HL export from the ER. Whereas a significant proportion of human HL can attain activity and become secreted in the absence of glucose trimming and calnexin association, these interrelated processes are nevertheless essential for the expression of full HL activity.     

Effect of recombinant Mce4A protein of Mycobacterium bovis on expression of TNF-α, iNOS, IL-6, and IL-12 in bovine alveolar macrophages

CD38 is a type II transmembrane protein with 25% of its molecular mass consisting of glycosyl moieties. It has long been predicted that the carbohydrate moieties of glycoproteins play important roles in the physical function and structural stability of the proteins on cell surfaces. To determine the structural/functional significance of glycosylation of the human CD38, the four potential N-linked glycosylation sites asparagine residues, N100, N164, N209 and N219 were mutated. The mutant (CD38mu) and wild-type (CD38wt) were expressed separately in Escherichia coli, HeLa, and MCF-7 cells. SDS-polyacrylamide gel electrophoresis under reducing conditions and western blotting indicated that the molecular mass of the CD38wt is 45 kDa, and that of the CD38mu is 34 kDa in HeLa cells. Importantly, the CD38mu protein expressed in HeLa cells, showed the high molecular weight oligomers in addition to the 34 kDa monomeric form. Similarly, in E. coli, the CD38wt formed dimers and other oligomers besides the monomeric form. Moreover, MCF-7 cells stably transfected with CD38wt cDNA, also revealed the presence of cross-linked oligomers when treated with a N-linked glycosylation inhibitor tunicamycin (TM). These results suggested that the N-linked glycosylation of CD38 plays a crucial role in the structure stability by preventing the formation inter-molecular cross-links. In addition, immunostaining, enzyme activity (cyclase), and western blotting data revealed that the glycosylation of human CD38 protein is not required for its localization to the cell membrane.     

The effect of N-linked glycosylation on the substrate preferences of UDP glucuronosyltransferases

The presence of potential N-linked glycosylation sites (Asn-X-Ser/Thr) in two forms of UDP glucuronosyltransferase, designated UDPGTr-2 and UDPGTr-4, has been deduced from cDNA sequence data. These forms were glycosylated when synthesized from expression vectors transfected into COS cells and were converted to faster migrating species on SDS polyacrylamide gels when treated with endoglycosidase H. The role of glycosylation was investigated by determining the substrate specificities and stabilities of the glycosylated enzymes and their unglycosylated variants which were synthesized in the presence of tunicamycin. Analysis of the activities towards 13 different aglycones showed that the glycosyl moiety was not essential for catalytic activity and had no effect on the substrate preference of each form. The stabilities of the proteins were not adversely affected by the absence of this posttranslational modification. A possible effect of N-linked oligosaccharides on the catalytic properties of these two forms of UDP glucuronosyltransferase is discussed.     

Isolation of a cDNA encoding the human CD38 (T10) molecule, a cell surface glycoprotein with an unusual discontinuous pattern of expression during lymphocyte differentiation

A cDNA clone encoding the human lymphocyte differentiation Ag CD38 was isolated from a mixture of four different lymphocyte CDNA libraries expressed transiently in COS cells and screened by panning with mAb. Transfected COS cells expressed a surface protein of Mr 46,000 that was similar to the native CD38 molecule expressed on the B cell line Daudi and the T cell leukemia HPB-ALL and which was recognized by each of the CD38 specific mAb HIT-2, T16, T168, HB7, 5D2, ICO-18, and ICO-20. The CD38 cDNA sequence predicts an unusual 30-kDa polypeptide with a short N-terminal cytoplasmic tail, and a carboxyl-terminal extracellular domain carrying the four potential N-linked glycosylation sites. The absence of significant homology with other known surface Ag including members of the Ig superfamily ruled out the possibility that CD38 was the human homologue of the murine Qa2 molecule as has been suggested previously. PvuII digests of human genomic DNA revealed a polymorphism linked to the CD38 gene.     

Common Human 5′ Dopamine Transporter (SLC6A3) Haplotypes Yield Varying Expression Levels In Vivo

1. Individuals display significant differences in their levels of expression of the dopamine transporter (DAT; SLC6A3). These differences in DAT are strong candidates to contribute to individual differences in motor, mnemonic and reward functions. To identify “cis”-acting genetic mechanisms for these individual differences, we have sought variants in 5′ aspects of the human DAT gene and identified the haplotypes that these variants define.2. We report (i) significant relationships between 5′ DAT haplotypes and human individual differences in ventral striatal DAT expression assessed in vivo using [¹¹C] cocaine PET and (ii) apparent confirmation of these results in studies of DAT expression in postmortem striatum using [³H] carboxyflurotropane binding.3. These observations support the idea that cis-acting variation in 5′ aspects of the human DAT/SLC6A3 locus contributes to individual differences in levels of DAT expression in vivo. 5′ DAT variation is thus a good candidate to contribute to individual differences in a number of human phenotypes.These authors contributed equally to this article     

Differences in the glycosylation of recombinant proteins expressed in HEK and CHO cells

Glycosylation is one of the most common posttranslational modifications of proteins. It has important roles for protein structure, stability and functions. In vivo the glycostructures influence pharmacokinetics and immunogenecity. It is well known that significant differences in glycosylation and glycostructures exist between recombinant proteins expressed in mammalian, yeast and insect cells. However, differences in protein glycosylation between different mammalian cell lines are much less well known. In order to examine differences in glycosylation in mammalian cells we have expressed 12 proteins in the two commonly used cell lines HEK and CHO. The cells were transiently transfected, and the expressed proteins were purified. To identify differences in glycosylation the proteins were analyzed on SDS-PAGE, isoelectric focusing (IEF), mass spectrometry and released glycans on capillary gel electrophoresis (CGE-LIF). For all proteins significant differences in the glycosylation were detected. The proteins migrated differently on SDS-PAGE, had different isoform patterns on IEF, showed different mass peak distributions on mass spectrometry and showed differences in the glycostructures detected in CGE. In order to verify that differences detected were attributed to glycosylation the proteins were treated with deglycosylating enzymes. Although, culture conditions induced minor changes in the glycosylation the major differences were between the two cell lines.     

Trafficking of dopamine transporters in psychostimulant actions

Brain dopamine (DA) plays a pivotal role in drug addiction. Since the plasma membrane DA transporter (DAT) is critical for terminating DA neurotransmission, it is important to understand how DATs are regulated and this regulation impacts drug addiction. The number of cell surface DATs is controlled by constitutive and regulated endocytic trafficking. Psychostimulants impact this trafficking. Amphetamines, DAT substrates, cause rapid up-regulation and slower down-regulation of DAT whereas cocaine, a DAT inhibitor, increases surface DATs. Recent reports have begun to elucidate the molecular mechanisms of these psychostimulant effects and link changes in DAT trafficking to psychostimulant-induced reward/reinforcement in animal models.     

Cloning and expression of a cDNA coding for a rat liver plasma membrane ecto-ATPase. The primary structure of the ecto-ATPase is similar to that of the human biliary glycoprotein I

The amino acid sequence of the ecto-ATPase from rat liver was deduced from analysis of cDNA clones and a genomic clone. Immunoblots with antibodies raised against a peptide sequence deduced from the cDNA sequence indicated that the determined amino acid sequence is that of the ecto-ATPase. The deduced sequence predicts a 519-amino acid protein with a calculated molecular mass of 57,388 daltons. There are 16 potential asparagine-linked glycosylation sites in the protein. Hydropathy analysis of the deduced amino acid sequence indicates that the protein has two hydrophobic stretches. One is located at the N-terminal and the other is near the C-terminal end. A full-length clone encoding the ecto-ATPase was expressed transiently in mouse L cells and human HeLa cells. The cell lysate from the transfected cells contained immunoreactive ecto-ATPase and Ca2+-stimulated ATPase activities. The expressed protein is glycosylated and has an apparent molecular weight (100,000) similar to that of the rat liver plasma membrane ecto-ATPase.     

Localization of cocaine analog [125I]RTI 82 irreversible binding to transmembrane domain 6 of the dopamine transporter

The site of cocaine binding on the dopamine transporter (DAT) was investigated using the photoactivatable irreversible cocaine analog [125I]3beta-(p-chlorophenyl)tropane-2beta-carboxylic acid, 4'-azido-3'-iodophenylethyl ester ([125I]RTI 82). The incorporation site of this compound was mapped to transmembrane domains (TMs) 4-6 using epitope-specific immunoprecipitation of trypsin fragments and further localized using cyanogen bromide (CNBr), which hydrolyzes proteins on the C-terminal side of methionine residues. CNBr hydrolysis of [125I]RTI 82-labeled rat striatal and expressed human DATs produced fragments of approximately 5-10 kDa consistent with labeling between Met(271/272) or Met(290) in TM5 to Met(370/371) in TM7. To further define the incorporation site, substitution mutations were made that removed endogenous methionines and inserted exogenous methionines in combinations that would generate labeled CNBr fragments of distinct masses depending on the labeling site. The results obtained were consistent with the presence of TM6 but not TMs 4, 5, or 7 in the labeled fragments, with additional support for these conclusions obtained by epitope-specific immunoprecipitation and secondary digestion of CNBr fragments with endoproteinase Lys-C. The final localization of [125I]RTI 82 incorporation to rat DAT Met(290)-Lys(336) and human DAT I291M to R344M provides positive evidence for the proximity of cocaine binding to TM6. Residues in and near DAT TM6 regulate transport and transport-dependent conformational states, and TM6 forms part of the substrate permeation pathway in the homologous Aquifex aeolicus leucine transporter. Cocaine binding near TM6 may thus overlap the dopamine translocation pathway and function to inhibit TM6 structural rearrangements necessary for transport.     

Rapid substrate-induced down-regulation in function and surface localization of dopamine transporters: rat dorsal striatum versus nucleus accumbens

The dopamine transporter (DAT) substrates dopamine, d-amphetamine (AMPH), and methamphetamine are known to rapidly and transiently reduce DAT activity and/or surface expression in dorsal striatum and heterologous expression systems. We sought to determine if similar substrate-induced regulation of DATs occurs in rat nucleus accumbens. In dorsal striatum synaptosomes, brief (15-min) in vitro substrate pre-exposure markedly decreased maximal [³H]dopamine uptake velocity whereas identical substrate pre-exposure in nucleus accumbens synaptosomes produced a smaller, non-significant reduction. However, 45 min after systemic AMPH administration, maximal ex vivo [³H]dopamine uptake velocity was significantly reduced in both brain regions. Protein kinase C inhibition blocked AMPH's down-regulation of DAT activity. DAT synaptosomal surface expression was not modified following either the brief in vitro or in vivo AMPH pre-exposure but was reduced after a longer (1-h) in vitro pre-exposure in both brain regions. Together, our findings suggest that relatively brief substrate exposure results in greater down-regulation of DAT activity in dorsal striatum than in nucleus accumbens. Moreover, exposure to AMPH appears to regulate striatal DATs in a biphasic manner, with an initial protein kinase C-dependent decrease in DAT-mediated uptake velocity and then, with longer exposure, a reduction in DAT surface expression.     

Active human erythropoietin expressed in insect cells using a baculovirus vector: A role for N-linked oligosaccharide

Biologically active recombinant human erythropoietin has been expressed at high levels in an insect cell background. Expression involved the preparation of a human erythropoietin cDNA, the transfer of this cDNA to the Autographa californica nuclear polyhedrosis virus (AcNPV) genome under the polyhedrin gene promoter, and the subsequent infection of Spodoptera frugiperda cells with recombinant AcNPV. Erythropoietin cDNA was prepared through the expression of the human erythropoietin gene in COS cells using pSV2 and the construction of a COS cell cDNA library in bacteriophage Lambda GT10. Prior to transfer to the AcNPV genome, erythropoietin cDNA isolated from this library was modified at the 3′-terminus in order to replace genomic erythropoietin for SV40 cDNA derived from pSV2. Transfer of this cDNA to AcNPV and the infection of S. frugiperda cells with cloned recombinant virus led to the secretion of erythropoietin: based on bioassay, rates of hormone secretion (over 40 U/ml per h) were 50-fold greater than observed for COS cells. The purified recombinant product possessed full biological activity (at least 200000 U/mg), but was of lower M_r (23000) than human erythropoietin produced in COS cells (30000) or purified from urine (30000 to 38000). This difference was attributed to the glycosylation of erythropoietin in S. frugiperda cells with oligosaccharides of only limited size. Further removal of N-linked oligosac-charides from this M_r 23000 hormone using N-Glycanase yielded an apo-erythropoietin (M_r 18000) which possessed substantially reduced biological activity. These results indicate that glycosylation, but not the normal processing of oligosaccharides to complex types, is required for the full hormonal activity of human erythropoietin during red cell development.     

Cloning and characterization of human Lnk, an adaptor protein with pleckstrin homology and Src homology 2 domains that can inhibit T cell activation

Lnk was originally cloned from a rat lymph node cDNA library and shown to participate in T cell signaling. Human Lnk (hLnk) was cloned by screening a Jurkat cell cDNA library. hLnk has a calculated molecular mass of 63 kDa, and its deduced amino acid sequence indicates the presence of an N-terminal proline-rich region, a pleckstrin homology domain, and a Src homology 2 domain. When expressed in COS cells, hLnk migrates with an apparent molecular mass of 75 kDa. Confocal fluorescence microscope analysis indicates that in COS cells transfected with an expression vector encoding a chimeric Lnk-green fluorescent protein, hLnk is found at the juxtanuclear compartment and also appears to be localized at the plasma membrane. Lnk is tyrosine-phosphorylated by p56lck. Following phosphorylation, p56lck binds to tyrosine-phosphorylated hLnk through its Src homology 2 domain. In COS cells cotransfected with hLnk, p56lck, and CD8-zeta, hLnk associated with tyrosine-phosphorylated TCR zeta-chain through its Src homology 2 domain. The overexpression of Lnk in Jurkat cells led to an inhibition of anti-CD3 mediated NF-AT-Luc activation. Our study reveals a potentially new mechanism of T cell-negative regulation.