Experimental data supporting the expression of the highly conserved GnRH-II in the brain and pituitary gland of rats

The second GnRH form, originally identified in chickens (cGnRH-II or GnRH-II), is the most ubiquitous peptide of the GnRH neuropeptide family, being present from jawed fish to human beings. However, the presence of GnRH-II in such an important experimental model as the rat is still an object of discussion. Here we present chromatographic, immunologic and biologic activity evidence supporting the expression of GnRH-II in the rat. Olfactory bulb, hypothalamus, remnant brain and anterior pituitary from a pool of 50 female adult rats were extracted and subjected to RP-HPLC on a C-18 column. The fractions were collected and evaluated by using two different RIA systems, specific for GnRH-I and GnRH-II respectively. Under these conditions the GnRH-I standard eluted in fraction 21 (f21) was only detected with the GnRH-I RIA system, whereas the GnRH-II standard was only detected in the fraction 27 (f27) by using a GnRH-II RIA system. In the olfactory bulbs extract, the fractions analyzed by the GnRH-I RIA systems showed a single peak in f21, whereas by using the GnRH-II RIA system a single peak at f27 was observed. In the hypothalamus GnRH-I was detected in f21 meanwhile GnRH-II could not be detected. When the remnant brain and pituitary gland extracts were analyzed, both GnRH forms were detected. To the best of our knowledge, this is the first report concerning GnRH-II detection in a mammalian pituitary. Serial dilutions of f27 and GnRH-II presented similar displacement of radioiodinated-GnRH-II, demonstrating that both molecules share immunological properties. Moreover, after 60 min stimulation, both f27 and GnRH-II had similar LH and FSH releasing activity in 12 day-old rat pituitary primary cell cultures. However, we failed to characterize the GnRH-II gene in this model. These results provide strong evidence for the expression of GnRH-II in the rat brain and pituitary gland.     

GnRH-II analogs for selective activation and inhibition of non-mammalian and type-II mammalian GnRH receptors

Recently, we identified three types of non-mammalian gonadotropin-releasing hormone receptors (GnRHR) in the bullfrog (designated bfGnRHR-1-3), and a mammalian type-II GnRHR in green monkey cell lines (denoted gmGnRHR-2). All these receptors responded better to GnRH-II than GnRH-I, while mammalian type-I GnRHR showed greater sensitivity to GnRH-I than GnRH-II. In the present study, we designed new GnRH-II analogs and examined whether they activated or inhibited non-mammalian and mammalian type-II GnRHRs. [D-Ala6]GnRH-II, with D-Ala substituted for Gly6 in GnRH-II, increased inositol phosphate (IP) production in cells stably expressing non-mammalian GnRHRs more effectively than native GnRH-II. However, it exhibited lower activity for mammalian type-I GnRHR than GnRH-I itself. Trptorelix-1, a GnRH-II antagonist, inhibited GnRH-induced IP production in cells expressing non-mammalian GnRHRs more effectively than Cetrorelix, a GnRH-I antagonist. Trptorelix-1, however, had lower potency for mammalian type-I GnRHR than Cetrorelix. Ligand-receptor binding assays revealed that [D-Ala6]GnRH-II and Trptorelix-1 have higher affinities for non-mammalian GnRHRs but lower affinities for mammalian type-I GnRHR than GnRH-II and Cetrorelix, respectively. Moreover, [D-Ala6]GnRH-II and Trptorelix-1 had a higher affinity for gmGnRHR-2 than GnRH-II and Cetrorelix, respectively. These results indicate that [D-Ala6]GnRH-II and Trptorelix-1 are highly effective agonist and antagonist, respectively, for non-mammalian and type-II mammalian GnRHRs.     

Mast cells in the rat brain synthesize gonadotropin-releasing hormone

Mast cells occur in the brain and their number changes with reproductive status. While it has been suggested that brain mast cells contain the mammalian hypothalamic form of gonadotropin-releasing hormone (GnRH-I), it is not known whether mast cells synthesize GnRH-I de novo. In the present study, mast cells in the rat thalamus were immunoreactive to antisera generated against GnRH-I and the GnRH-I associated peptide (GAP); mast cell identity was confirmed by the presence of heparin, a molecule specific to mast cells, or serotonin. To test whether mast cells synthesize GnRH-I mRNA, in situ hybridization was performed using a GnRH-I cRNA probe, and the signal was identified as being within mast cells by the binding of avidin to heparin. GnRH-I mRNA was also found, using RT-PCR, in mast cells isolated from the peritoneal cavity. Given the function of GnRH-I in the regulation of reproduction, changes in the population of brain GnRH-I mast cells were investigated. While housing males with sexually receptive females for 2 h or 5 days resulted in a significant increase in the number of brain mast cells, the proportion of mast cells positive for GnRH-I was similar to that in males housed with a familiar male. These findings represent the first report showing that mast cells synthesize GnRH-I and that the mast cell increase seen in a reproductive context is the result of a parallel increase in GnRH-I positive and non-GnRH-I positive mast cells.     

Structure-function studies of five natural, including catfish and dogfish, gonadotropin-releasing hormones and eight analogs on reproduction in Thai catfish (Clarias macrocephalus).

Five distinct forms of gonadotropin-releasing hormone (GnRH) and their analogs, six of which are newly designed, were used to study reproduction in Thai catfish, Clarias macrocephalus. Determination was made for the percentage of fish that ovulated within 16-18 h; the percentage of eggs fertilized; and the percentage of larva that hatched and survived for 7 days. The results show, firstly, that natural chicken GnRH-II, which is identical with catfish GnRH-II, was significantly more effective at a dose of 300 micrograms/kg than the control injection for the induction of ovulation. Dogfish GnRH at the same dose was also significantly more effective than the control, but was not significantly different from chicken (catfish) GnRH-II for ovulation induction. The novel catfish GnRH-I, mammalian GnRH and salmon GnRH were not effective at 100, 150 or 300 micrograms/kg in Thai catfish. Secondly, 5 of 8 analogs of GnRH at a dose of 20 micrograms/kg resulted in a significantly higher percentage of ovulating fish compared with the control fish. Among these five analogs, the most effective were the two analog forms of chicken GnRH-II (D-Arg6,Pro9 NEt and D-Nal6,Pro9 NEt), followed by the salmon GnRH analog (D-Arg6,Pro9 NEt), a dogfish GnRH analog (D-Arg6,Pro9 NEt) and the mammalian GnRH analog (D-Ala6,Pro9 NEt). Not significantly different from the controls were the two catfish GnRH-I analogs and one of the dogfish (D-Nal6,Pro9 NEt) analogs. The six new analogs had not been previously tested in any animal. Thirdly, the number of fish ovulating was the same whether GnRH was administered in one or two injections.     

Second form of gonadotropin-releasing hormone in mouse: immunocytochemistry reveals hippocampal and periventricular distribution

Hypothalamic GnRH (GnRH-I) is known and named for its role in regulating reproductive function in vertebrates by controlling release of gonadotropins from the pituitary. However, another form of GnRH of unknown function (pGlu-His-Trp-Ser-His-Gly-Trp-Tyr-Pro-Gly; GnRH-II) is expressed in the mesencephalon of all vertebrate classes except jawless fish. Here we show with immunocytochemical staining that the GnRH-II peptide is localized to the mouse midbrain as in other vertebrates, as well as in cells surrounding the ventricles and in cells adjacent to the hippocampus. Staining of adjacent sections using GnRH-I antibody revealed that the distribution of GnRH-I does not overlap with that of GnRH-II.     

Gonadotropin-releasing hormone (GnRH)-I and GnRH-II induce cell growth inhibition in human endometrial cancer cells: Involvement of integrin beta3 and focal adhesion kinase

Endometrial carcinoma is the most common neoplasm of the female genital tract, accounting for nearly one half of all gynecologic cancers in the Western world. Although intensive research on pathological phenomena of endometrial cancer is currently going on, but exact cause and biological aspects of this disease are not well described yet. In addition to well-documented roles of gonadotropin-releasing hormone (GnRH) in hypopituitary ovarian (HPO) axis, the agonistic or antagonistic analogs (or both) of GnRH have been shown to inhibit the proliferation of a variety of human gynecologic cancers. Thus, in the present study, we further examined the possibility that GnRH induces integrin beta3 and activation of focal adhesion kinase (FAK) through mitogen-activated protein kinases (MAPKs), ERK1/2 and p38, to inhibit the growth of HEC1A endometrial cancer cell line. As a result, both GnRH-I and GnRH-II resulted in a significant increase in integrin beta3 expression and evoked the activation of FAK in a time-dependent manner in these cells. In addition, these analogs induced an activation of ERK1/2 and p38 MAPK in a time-dependent manner as downstream pathways of FAK. It appears that GnRH-II has much greater effect on the activation of FAK, ERK1/2 and p38 compared to GnRH-I in these cells. Further, we demonstrated that the growth inhibition of HEC1A cells by GnRH-I or GnRH-II is involved in the activation of integrin-FAK and ERK1/2 and p38 MAPK pathways. Taken together, these results suggest that GnRH may be involved in the inhibition of endometrial cancer cell growth via activation of integrin beta3 and FAK as a direct effect. This knowledge could contribute to a better understanding of the mechanisms implicated in the therapeutic action of GnRH and its biomedical application for the treatment against endometrial cancer.     

Evidence for different gonadotropin-releasing hormone response sites in rat ovarian and pituitary cells

The participation of type I GnRH receptor (GnRH-R) on GnRH-II-induced gonadotropin secretion in rat pituitary cells was investigated. Furthermore, we extended the study of GnRH-II action to ovarian cells. The GnRH-II was able to mobilize inositol triphosphate (IP(3)) and to induce LH and FSH release in a dose-dependent manner in pituitary cells and in a GnRH-I-like manner. The GnRH-analog 135-18 (agonist for type II GnRH-R and antagonist for type I GnRH-R) was unable to elicit any cellular response tested in these pituitary cells. The GnRH-II responses were blocked by the type I GnRH-R-antagonists CRX or 135-18, suggesting that these effects were mediated by the type I GnRH-R. In contrast to pituitary cells, GnRH-I, but not GnRH-II, elicited an IP(3) response in superovulated ovarian cells; 135-18 also had no effect. However, GnRH-II as well as GnRH-I presented antiproliferative effects on these cells. Surprisingly, 135-18 had stronger antiproliferative effects than either GnRH peptide. The 135-18 analog, but not GnRH-I or GnRH-II, increased progesterone secretion in superovulated ovarian cells. These results strongly suggest that GnRH-II is able to stimulate rat pituitary cells through the type I GnRH-R, with no evidence for the presence of type II GnRH-R. On the other hand, our results indicate a putative GnRH-R in superovulated ovarian cells with response characteristics that differ from those of the GnRH-R in the pituitary.     

37-kDa laminin receptor precursor mediates GnRH-II-induced MMP-2 expression and invasiveness in ovarian cancer cells

GnRH-II enhances ovarian cancer cell invasion in an autocrine manner. We have now found that GnRH-II increases 37-kDa laminin receptor precursor (LRP) production in GnRH receptor (GnRHR)-positive OVCAR-3 and CaOV-3 ovarian cancer cells, while small interfering RNA (siRNA)-mediated depletion of GnRH-II or GnRHR mRNA abrogates this. The invasiveness of ovarian cancer cells is also reduced >85% by siRNA-mediated knockdown of LRP levels and >50% by pretreatment of Matrigel with a synthetic peptide that blocks interactions between laminin and the 67-kDa nonintegrin laminin receptor which comprises two LRP subunits. Conversely, overexpressing LRP in CaOV-3 cells increases their invasiveness 5-fold, while overexpressing LRP with a nonfunctional laminin-binding site does not. Depletion of LRP by siRNA treatment reduces CaOV-3 cell attachment to laminin-coated plates by ～80% but only reduces their binding to Matrigel by ～20%. Thus, while LRP influences CaOV-3 cell adhesion to laminin, LRP must act in other ways to enhance invasion. Matrix metalloproteinases (MMPs) are key mediators of invasion, and LRP siRNA treatment of OVCAR-3 and CaOV-3 cells inhibits MMP-2 but not MMP-9 mRNA levels. Overexpressing LRP in these cells increases MMP-2 production specifically, while a laminin-binding deficient LRP does not. Importantly, LRP siRNA treatment abolishes GnRH-II-induced MMP-2 production, and invasion in OVCAR-3 and CaOV-3 cells, which was also seen after MMP-2 siRNA treatment. These results suggest that GnRH-II-induced LRP expression increases the amount of the 67-kDa nonintegrin laminin receptor, which appears to interact with laminin in the extracellular matrix to promote MMP-2 expression and enhance ovarian cancer cell invasion.     

Cloning and functional analysis of the proximal promoter region of the three GnRH genes from the silver sea bream (Sparus sarba)

Gonadotropin-releasing hormone (GnRH) is a neuropeptide that plays a major role in releasing pituitary gonadotropin and controlling vertebrate reproduction. In this study, three GnRH cDNAs, GnRH-I (sbGnRH; 348 bp), GnRH-II (cGnRH-II; 557 bp), and GnRH-III (sGnRH; 483 bp), were cloned from the brain of the silver sea bream (Sparus sarba). In order to understand how the expression of the GnRH isoforms was regulated in the brain, the promoter of each gene was cloned and analyzed. We found regulatory motifs in the promoters that were conserved in the GnRH promoters of tilapia and zebrafish, suggesting that these motifs play a critical role in GnRH regulation. We performed functional analyses and examined tissue-specific expression for each GnRH promoter using EGFP reporter fusions in zebrafish. The GnRH-I promoter was active in the forebrain area, including the olfactory bulb-terminal nerve area and peripheral preoptic areas; the GnRH-II promoter was active in the midbrain; and the GnRH-III promoter was active in the olfactory bulb. These results show that the GnRH promoters of the silver sea bream GnRH genes exhibit tissue-specific activity.     

Involvement of amino acids flanking Glu7.32 of the gonadotropin-releasing hormone receptor in the selectivity of antagonists

The Glu/Asp(7.32) residue in extracellular loop 3 of the mammalian type-I gonadotropin-releasing hormone receptor (GnRHR) interacts with Arg(8) of GnRH-I, conferring preferential ligand selectivity for GnRH-I over GnRH-II. Previously, we demonstrated that the residues (Ser and Pro) flanking Glu/Asp(7.32) also play a role in the differential agonist selectivity of mammalian and non-mammalian GnRHRs. In this study, we examined the differential antagonist selectivity of wild type and mutant GnRHRs in which the Ser and Pro residues were changed. Cetrorelix, a GnRH-I antagonist, and Trptorelix-2, a GnRH-II antagonist, exhibited high selectivity for mammalian type-I and non-mammalian GnRHRs, respectively. The inhibitory activities of the antagonists were dependent on agonist concentration and subtype. Rat GnRHR in which the Ser-Glu-Pro (SEP) motif was changed to Pro-Glu-Val (PEV) or Pro-Glu-Ser (PES) had increased sensitivity to Trptorelix-2 but decreased sensitivity to Cetrorelix. Mutant bullfrog GnRHR-1 with the SEP motif had the reverse antagonist selectivity, with reduced sensitivity to Trptorelix-2 but increased sensitivity to Cetrorelix. These findings indicate that the residues flanking Glu(7.32) are important for antagonist as well as agonist selectivity.     

Expression of chemokine receptors by lung T cells from normal and asthmatic subjects

The lung is an important tertiary lymphoid organ with constant trafficking of T cells through the lung in both health and disease. T cell migration is controlled by a combination of adhesion receptors and chemokines expressed on vascular endothelium and in the tissue, often in an organ-specific manner. This leads to selective accumulation of different T cell subsets, a process called lymphocyte homing. There is evidence for a distinct lung-homing pathway, but no specific lung-homing receptors have been described. We analyzed the chemokine receptor profile of lung T cells to determine the extent to which lung T cells shared homing pathways with other organs such as the gut and skin. In addition, we compared expression of receptors in normal and asthmatic individuals to determine whether different pathways were used in health and disease. We observed that lung T cells expressed a profile of chemokine and adhesion receptors distinct from that of gut- and skin-homing T cells although no chemokine receptor specific for the lung was found. In particular, lung T cells expressed CCR5 and CXCR3, but not CCR9 or cutaneous lymphocyte Ag, and only low levels of CCR4 and alpha(4)beta(7). No differences were observed between lung T cells from normal vs asthmatic subjects. This study provides added support for the concept of a lung-homing pathway separate from other mucosal organs such as the gut and suggests that the chemokine pathways that control T cell migration in normal homeostasis and Th2-type inflammatory responses are similar.     

Laminin-211 controls thymocyte--thymic epithelial cell interactions

Thymocyte differentiation occurs within the thymic microenvironment, consisting of distinct cell types and extracellular matrix (ECM) elements. One of these ECM proteins is laminin. Previous experiments showed that laminin mediates interactions between thymocytes and thymic epithelial cells (TEC) in mice. Since, laminin comprises a family of related isoforms, we searched for laminin isoform expression in the human thymus. We found constitutive gene expression of various laminin chains in TEC preparations, comprising laminin-111 and laminin-211 isoforms. Immunocytochemistry revealed a selective laminin-211 distribution in the thymic lobules. In vitro functional assays revealed that laminin-211 enhances TEC/thymocyte adhesion and thymocyte release from thymic nurse cells, as well as the reconstitution of these complexes. Conversely, these interactions are blocked by monoclonal antibodies specific for laminin-211 and the laminin receptor VLA-6. Our results reinforce the notion that distinct laminin isoforms in the human thymus are relevant for lymphoepithelial interactions.     

Endothelial cell laminin isoforms, laminins 8 and 10, play decisive roles in T cell recruitment across the blood-brain barrier in experimental autoimmune encephalomyelitis

An active involvement of blood-brain barrier endothelial cell basement membranes in development of inflammatory lesions in the central nervous system (CNS) has not been considered to date. Here we investigated the molecular composition and possible function of the extracellular matrix encountered by extravasating T lymphocytes during experimental autoimmune encephalomyelitis (EAE).Endothelial basement membranes contained laminin 8 (alpha4beta1gamma1) and/or 10 (alpha5beta1gamma1) and their expression was influenced by proinflammatory cytokines or angiostatic agents. T cells emigrating into the CNS during EAE encountered two biochemically distinct basement membranes, the endothelial (containing laminins 8 and 10) and the parenchymal (containing laminins 1 and 2) basement membranes. However, inflammatory cuffs occurred exclusively around endothelial basement membranes containing laminin 8, whereas in the presence of laminin 10 no infiltration was detectable. In vitro assays using encephalitogenic T cell lines revealed adhesion to laminins 8 and 10, whereas binding to laminins 1 and 2 could not be induced. Downregulation of integrin alpha6 on cerebral endothelium at sites of T cell infiltration, plus a high turnover of laminin 8 at these sites, suggested two possible roles for laminin 8 in the endothelial basement membrane: one at the level of the endothelial cells resulting in reduced adhesion and, thereby, increased penetrability of the monolayer; and secondly at the level of the T cells providing direct signals to the transmigrating cells.     

Bridging structure with function: structural, regulatory, and developmental role of laminins

The basement membrane is a highly intricate and organized portion of the extracellular matrix that interfaces with a variety of cell types including epithelial, endothelial, muscle, nerve, and fat cells. The laminin family of glycoproteins is a major constituent of the basement membrane. The 16 known laminin isoforms are formed from combinations of alpha, beta, and gamma chains, with each chain containing specific domains capable of interacting with cellular receptors such as integrins and other extracellular ligands. In addition to its role in the assembly and architectural integrity of the basement membrane, laminins interact with cells to influence proliferation, differentiation, adhesion, and migration, processes activated in normal and pathologic states. In vitro these functions are regulated by the post-translational modifications of the individual laminin chains. In vivo laminin knockout mouse studies have been particularly instructive in defining the function of specific laminins in mammalian development and have also highlighted its role as a key component of the basement membrane. In this review, we will define how laminin structure complements function and explore its role in both normal and pathologic processes.     

Integrin recognition of different cell-binding fragments of laminin (P1, E3, E8) and evidence that alpha 6 beta 1 but not alpha 6 beta 4 functions as a major receptor for fragment E8

The involvement of integrins in mediating interaction of cells to well-characterized proteolytic fragments (P1, E3, and E8) of laminin was assessed by antibody blocking studies. Cell adhesion to fragment P1 was affected by mAbs against the integrin beta 1 and beta 3 subunits and furthermore could be prevented completely by a synthetic peptide containing the Arg-Gly-Asp sequence. Because the beta 3 antibody-sensitive cell lines expressed the vitronectin receptor (alpha v beta 3) at high levels, the involvement of this receptor in cell adhesion to P1 is strongly suggested. Integrin-mediated cell adhesion to E3 is of low affinity and was inhibited by antibodies against the integrin beta 1 subunit. In contrast, adhesion of some cell types to E3 was not or only partially sensitive to inhibition by anti-integrin subunit antibodies. Cell adhesion to E8 was blocked completed by integrin alpha 6 or beta 1 antibodies. The alpha 6-specific antibody did not inhibit cell adhesion to E3 or P1. Furthermore, the antibody only blocked adhesion to laminin of those cells that adhered exclusively to the E8 fragment. In addition, expression of alpha 6 beta 1 was closely correlated with the ability of cells to bind to the E8 fragment of laminin. These results indicate that the alpha 6 beta 1 integrin is a specific receptor for the E8 fragment of laminin. Many cell types expressed, instead of or in addition to alpha 6 beta 1 the recently described integrin alpha 6 beta 4. Although the ligand of alpha 6 beta 4 was not identified, it must be different from that of alpha 6 beta 1, because cells that express alpha 6 beta 4, but not alpha 6 beta 1, do not adhere to E8, and cell adhesion to E8 was specifically blocked by beta 1 specific antibodies. In conclusion, the data indicate that distinct integrin receptors belonging to the beta 1 or beta 3 subfamily are involved in adhesion of cells to the various laminin fragments. Adhesion to E3 may also be brought about by other receptor molecules, possibly proteoglycans, not belonging to the integrin family.     

The activation dependent adhesion of macrophages to laminin involves cytoskeletal anchoring and phosphorylation of the alpha 6 beta 1 integrin

Macrophages require activation with either PMA (Mercurio, A. M., and L. M. Shaw. 1988. J. Cell Biol. 107:1873-1880) or interferon-gamma (Shaw, L. M., and A. M. Mercurio. 1989. J. Exp. Med. 169:303-308) to adhere to a laminin substratum. In the present study, we identified an integrin laminin receptor on macrophages and characterized cellular changes that occur in response to PMA activation that facilitate laminin adhesion. A monoclonal antibody (GoH3) that recognizes the integrin alpha 6 subunit (Sonnenberg, A., H. Janssen, F. Hogervorst, J. Calafat, and J. Hilgers. 1987. J. Biol. Chem. 262:10376-10383) specifically inhibited adhesion to laminin-coated surfaces. This antibody precipitated an alpha 6 beta 1 heterodimer (Mr 130/110 kD) from 125I surface-labeled macrophages. The amount of radiolabeled receptor on the cell surface did not increase after PMA activation. Thus, the induction of laminin adhesion cannot be attributed to de novo or increased surface expression of alpha 6 beta 1. By initially removing the Triton X-100-soluble fraction of macrophages and then disrupting the remaining cytoskeletal framework, we observed that 75% of the alpha 6 beta 1 heterodimer on the cell surface is anchored to the cytoskeleton in macrophages that had adhered to a laminin substratum in response to PMA. Significant cytoskeletal anchoring of this receptor was not observed in macrophages that had adhered to fibronectin or tissue culture plastic, nor was it seen in nonadherent cells. PMA also induced phosphorylation of the cytoplasmic domain of the alpha 6 subunit, but not the beta 1 subunit. Phosphorylated alpha 6 was localized to the cytoskeletal fraction only in macrophages plated on a laminin substratum. In summary, our results support a mechanism for the regulation of macrophage adhesion to laminin that involves specific and dynamic matrix integrin-cytoskeletal interactions that may be facilitated by integrin phosphorylation.     

Human T cells express a functional ionotropic glutamate receptor GluR3, and glutamate by itself triggers integrin-mediated adhesion to laminin and fibronectin and chemotactic migration

T cells may encounter glutamate, the major excitatory neurotransmitter in the nervous system, when patrolling the brain and in glutamate-rich peripheral organs. Moreover, glutamate levels increase in the CNS in many pathological conditions in which T cells exert either beneficial or detrimental effects. We discovered that normal human T cells, human T leukemia cells, and mouse anti-myelin basic protein T cells express high levels of glutamate ion channel receptor (ionotropic) of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) subtype 3 (GluR3). The evidence for GluR3 on T cells includes GluR3-specific RT-PCR, Western blot, immunocytochemical staining and flow cytometry. Sequencing showed that the T cell-expressed GluR3 is identical with the brain GluR3. Glutamate (10 nM), in the absence of any additional molecule, triggered T cell function: integrin-mediated T cell adhesion to laminin and fibronectin, a function normally performed by activated T cells only. The effect of glutamate was mimicked by AMPA receptor-agonists and blocked specifically by the selective receptor-antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 6-nitro-7-sulfamoylbenzo[f]quinoxalin-2,3-dione (NBQX), and by relevant anti-integrin mAbs. Glutamate also increased the CXCR4-mediated T cell chemotactic migration toward the key chemokine CXCL12/stromal cell-derived factor-1. GluR3 expression on normal, cancer and autoimmune-associated T cells and the ability of glutamate to directly activate T cell function could be of substantial scientific and clinical importance to normal neuroimmune dialogues and to CNS diseases and injury, and especially to: 1) T cell transmigration to the CNS and patrolling in the brain, 2) T cell-mediated multiple sclerosis, and 3) autoimmune epilepsy, as neurotoxic anti-GluR3 Abs are found and suspected to cause/potentiate seizures and neuropathology in several types of human epilepsies. Thus far, GluR3 was found only on neurons and glia cells; our results reveal a novel peripheral source of this antigenic receptor.