Biomedical subjects

C Lluis

Publications and source records attributed to C Lluis.

At least 19 recordsLinked to original sources

PubMed2006

Intramembrane receptor-receptor interactions: a novel principle in molecular medicine.

In 1980/81 Agnati and Fuxe introduced the concept of intramembrane receptor-receptor interactions and presented the first experimental observations for their existence in crude membrane preparations. The second step was their introduction of the receptor mosaic hypothesis of the engram in 1982. The third step was their proposal that the existence of intramembrane receptor-receptor interactions made possible the integration of synaptic (WT) and extrasynaptic (VT) signals. With the discovery of the intramembrane receptor-receptor interactions with the likely formation of receptor aggregates of multiple receptors, so called receptor mosaics, the entire decoding process becomes a branched process already at the receptor level in the surface membrane. Recent developments indicate the relevance of cooperativity in intramembrane receptor-receptor interactions namely the presence of regulated cooperativity via receptor-receptor interactions in receptor mosaics (RM) built up of the same type of receptor (homo-oligomers) or of subtypes of the same receptor (RM type1). The receptor-receptor interactions will to a large extent determine the various conformational states of the receptors and their operation will be dependent on the receptor composition (stoichiometry), the spatial organization (topography) and order of receptor activation in the RM. The biochemical and functional integrative implications of the receptor-receptor interactions are outlined and long-lived heteromeric receptor complexes with frozen RM in various nerve cell systems may play an essential role in learning, memory and retrieval processes. Intramembrane receptor-receptor interactions in the brain have given rise to novel strategies for treatment of Parkinson's disease (A2A and mGluR5 receptor antagonists), schizophrenia (A2A and mGluR5 agonists) and depression (galanin receptor antagonists). The A2A/D2, A2A/D3 and A2A/mGluR5 heteromers and heteromeric complexes with their possible participation in different types of RM are described in detail, especially in the cortico-striatal glutamate synapse and its extrasynaptic components, together with a postulated existence of A2A/D4 heteromers. Finally, the impact of intramembrane receptor-receptor interactions in molecular medicine is discussed outside the brain with focus on the endocrine, the cardiovascular and the immune systems.

Animals↗

PubMed2006

Receptor-receptor interactions involving adenosine A1 or dopamine D1 receptors and accessory proteins.

The molecular basis for the known intramembrane receptor-receptor interactions among heptahelical receptors (G protein coupled receptors, GPCR) was postulated to be heteromerization based on receptor subtype specific interactions between different types of homomers of GPCR. Adenosine and dopamine receptors in the basal ganglia have been fundamental to demonstrate the existence of receptor heteromers and the functional consequences of such molecular interactions. The heterodimer is only one type of heteromeric complex and the evidence is equally compatible with the existence of higher order heteromeric complexes, where also adapter proteins such as homer proteins and scaffolding proteins can exist, assisting in the process of linking the GPCR and ion channel receptors together in a receptor mosaic that may have special integrative value and may constitute the molecular basis for learning and memory. Heteromerization of D(2) dopamine and A(2A) adenosine receptors is reviewed by Fuxe in another article in this special issue. Here, heteromerization between D(1) dopamine and A(1) adenosine receptors is reviewed. Heteromers formed by dopamine D(1) and D(2) receptors and by adenosine A(1) and A(2A) receptors also occur in striatal cells and open new perspectives to understand why two receptors with apparently opposite effects are expressed in the same neuron and in the nerve terminals. The role of accessory proteins also capable of interacting with receptor-receptor heteromers in regulating the traffic and the molecular physiology of these receptors is also discussed. Overall, the knowledge of the reason why such complex networks of receptor-receptor and receptor-protein interactions occur in striatal cells is crucial to develop new strategies to combat neurological and neuropsychiatric diseases.

Animals↗

PubMed2006

Heterodimeric adenosine receptors: a device to regulate neurotransmitter release.

Since 1990 it has been known that dimers are the basic functional form of nearly all G-protein-coupled receptors (GPCRs) and that homo- and heterodimerization may play a key role in correct receptor maturation and trafficking to the plasma membrane. Nevertheless, homo- and heterodimerization of GPCR has become a matter of debate especially in the search for the precise physiological meaning of this phenomenon. This article focuses on how heterodimerization of adenosine A1 and A2A receptors, which are coupled to apparently opposite signalling pathways, allows adenosine to exert a fine-tuning modulation of striatal glutamatergic neurotransmission, providing a switch mechanism by which low and high concentrations of adenosine inhibit and stimulate, respectively, glutamate release.

Adenosine↗

PubMed2005

CD26, adenosine deaminase, and adenosine receptors mediate costimulatory signals in the immunological synapse.

Adenosine deaminase (ADA), a protein whose deficit leads to severe combined immunodeficiency, binds to the cell surface by means of either CD26, A(1) adenosine receptors, or A(2B) adenosine receptors. The physiological role of these interactions is not well understood. Our results show that by a 3-fold reduction in the EC(50) for the antigen, ADA potentiated T cell proliferation in autologous cocultures with antigen-pulsed immature or mature dendritic cells. Costimulation was not due to the enzymatic activity but to the interaction of ADA-CD26 complexes in T cells with an ADA-anchoring protein in dendritic cells. From colocalization studies, it is deduced that ADA colocalizing with adenosine receptors on dendritic cells interact with CD26 expressed on lymphocytes. This costimulatory signal in the immunological synapse leads to a marked increase (3- to 34-fold) in the production of the T helper 1 and proimmflamatory cytokines IFN-gamma, TNF-alpha, and IL-6.

Adenosine Deaminase↗

PubMed2003

Receptor heteromerization in adenosine A2A receptor signaling: relevance for striatal function and Parkinson's disease.

Recently evidence has been presented that adenosine A2A and dopamine D2 receptors form functional heteromeric receptor complexes as demonstrated in human neuroblastoma cells and mouse fibroblast Ltk- cells. These A2A/D2 heteromeric receptor complexes undergo coaggregation, cointernalization, and codesensitization on D2 or A2A receptor agonist treatments and especially after combined agonist treatment. It is hypothesized that the A2A/D2 receptor heteromer represents the molecular basis for the antagonistic A2A/D2 receptor interactions demonstrated at the biochemical and behavioral levels. Functional heteromeric complexes between A2A and metabotropic glutamate 5 receptors (mGluR5) have also recently been demonstrated in HEK-293 cells and rat striatal membrane preparations. The A2A/mGluR5 receptor heteromer may account for the synergism found after combined agonist treatments demonstrated in different in vitro and in vivo models. D2, A2A, and mGluR5 receptors are found together in the dendritic spines of the striatopallidal GABA neurons. Therefore, possible D2/A2A/mGluR5 multimeric receptor complexes and the receptor interactions within them may have a major role in controlling the dorsal and ventral striatopallidal GABA neurons involved in Parkinson's disease and in schizophrenia and drug addiction, respectively.

Animals↗

PubMed2002

Interactions among adenosine deaminase, adenosine A(1) receptors and dopamine D(1) receptors in stably cotransfected fibroblast cells and neurons.

The role of adenosine deaminase in the interactions between adenosine A(1) and dopamine D(1) receptors was studied in a mouse fibroblast cell line stably cotransfected with human D(1) receptor and A(1) receptor cDNAs (A(1)D(1) cells). Confocal laser microscopy analysis showed a high degree of adenosine deaminase immunoreactivity on the membrane of the A(1)D(1) cells but not of the D(1) cells (only cotransfected with human D(1) receptor cDNAs). In double immunolabelling experiments in A(1)D(1) cells and cortical neurons a marked overlap in the distribution of the A(1) receptor and adenosine deaminase immunoreactivities and of the D(1) receptor and adenosine deaminase immunoreactivities was found. Quantitative analysis of A(1)D(1) cells showed that adenosine deaminase immunoreactivity to a large extent colocalizes with A(1) and D(1) receptor immunoreactivity, respectively. The A(1) receptor agonist caused in A(1)D(1) cells and in cortical neurons coaggregation of A(1) receptors and adenosine deaminase, and of D(1) receptors and adenosine deaminase. The A(1) receptor agonist-induced aggregation was blocked by R-deoxycoformycin, an irreversible adenosine deaminase inhibitor. The competitive binding experiments with the D(1) receptor antagonist [(3)H]SCH-23390 showed that the D(1) receptors had a better fit for two binding sites for dopamine, and treatment with the A(1) receptor agonist produced a disappearance of the high-affinity site for dopamine at the D(1) receptor. R-Deoxycoformycin treatment, which has previously been shown to block the interaction between adenosine deaminase and A(1) receptors, and which is crucial for the high-affinity state of the A(1) receptor, also blocked the A(1) receptor agonist-induced loss of high-affinity D(1) receptor binding. The conclusion of the present studies is that the high-affinity state of the A(1) receptor is essential for the A(1) receptor-mediated antagonistic modulation of D(1) receptors and for the A(1) receptor-induced coaggregates of A(1) and adenosine deaminase, and of D(1) and adenosine deaminase. Thus, the confocal experiments indicate that both A(1) and D(1) receptors form agonist-regulated clusters with adenosine deaminase, where the presence of a structurally intact adenosine deaminase bound to A(1) receptors is important for the A(1)-D(1) receptor-receptor interaction at the level of the D(1) receptor recognition.

2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-ben↗

PubMed2001

Metabotropic glutamate 1alpha and adenosine A1 receptors assemble into functionally interacting complexes.

Recently, evidence has emerged that seven transmembrane G protein-coupled receptors may be present as homo- and heteromers in the plasma membrane. Here we describe a new molecular and functional interaction between two functionally unrelated types of G protein-coupled receptors, namely the metabotropic glutamate type 1alpha (mGlu(1alpha) receptor) and the adenosine A1 receptors in cerebellum, primary cortical neurons, and heterologous transfected cells. Co-immunoprecipitation experiments showed a close and subtype-specific interaction between mGlu(1alpha) and A1 receptors in both rat cerebellar synaptosomes and co-transfected HEK-293 cells. By using transiently transfected HEK-293 cells a synergy between mGlu(1alpha) and A1 receptors in receptor-evoked [Ca(2+)](i) signaling has been shown. In primary cultures of cortical neurons we observed a high degree of co-localization of the two receptors, and excitotoxicity experiments in these cultures also indicate that mGlu(1alpha) and A1 receptors are functionally related. Our results provide a molecular basis for adenosine/glutamate receptors cross-talk and open new perspectives for the development of novel agents to treat neuropsychiatric disorders in which abnormal glutamatergic neurotransmission is involved.

Animals↗

PubMed2001

Comodulation of CXCR4 and CD26 in human lymphocytes.

We provide convergent and multiple evidence for a CD26/CXCR4 interaction. Thus, CD26 codistributes with CXCR4, and both coimmunoprecipitate from membranes of T (CD4(+)) and B (CD4(-)) cell lines. Upon induction with stromal cell-derived factor 1alpha (SDF-1alpha), CD26 is cointernalized with CXCR4. CXCR4-mediated down-regulation of CD26 is not induced by antagonists or human immunodeficiency virus (HIV)-1 gp120. SDF-1alpha-mediated down-regulation of CD26 is not blocked by pertussis toxin but does not occur in cells expressing mutant CXCR4 receptors unable to internalize. Codistribution and cointernalization also occurs in peripheral blood lymphocytes. Since CD26 is a cell surface endopeptidase that has the capacity to cleave SDF-1alpha, the CXCR4.CD26 complex is likely a functional unit in which CD26 may directly modulate SDF-1alpha-induced chemotaxis and antiviral capacity. CD26 anchors adenosine deaminase (ADA) to the lymphocyte cell surface, and this interaction is blocked by HIV-1 gp120. Here we demonstrate that gp120 interacts with CD26 and that gp120-mediated disruption of ADA/CD26 interaction is a consequence of a first interaction of gp120 with a domain different from the ADA binding site. SDF-1alpha and gp120 induce the appearance of pseudopodia in which CD26 and CXCR4 colocalize and in which ADA is not present. The physical association of CXCR4 and CD26, direct or part of a supramolecular structure, suggests a role on the function of the immune system and the pathophysiology of HIV infection.

Adenosine Deaminase↗

PubMed2001

Adenosine A2B receptors behave as an alternative anchoring protein for cell surface adenosine deaminase in lymphocytes and cultured cells.

Adenosine deaminase (ADA) is an enzyme of the purine metabolism that has been largely considered to be cytosolic. Recently, it has been demonstrated that the enzyme appears on the surface of lymphocytes where it interacts with the T-cell activation antigen CD26. ADA also appears on the surface of nonlymphoid cells anchored to adenosine A1 receptors. Here it is demonstrated that cell surface ADA in ADA+/CD26- T lymphocytes anchors to adenosine receptors of the A2B subtype (A2BR). An interaction between A2BR and cell surface ADA has been demonstrated in transfected Chinese hamster ovary cells and Jurkat J32 T lymphocytes. This has been proved by coimmunoprecipitation, binding of exogenous ADA to A2BR+ cells, and coimmunolocalization. The specificity of the interaction has also been demonstrated by the lack of interaction with other members of the G protein-coupled receptor superfamily. Binding of ADA to A2BR increases the affinity of the agonist 5'-N-ethylcarboxamidoadenosine and cAMP production. This effect occurs even when ADA devoid of enzyme activity is used. Therefore, in lymphocytes, cell surface ADA, apart from degrading extracellular adenosine, regulates those actions of adenosine that are mediated via adenosine receptors of the A2B subtype.

Adenosine Deaminase↗

PubMed2001

Cytokines regulate membrane adenosine deaminase on human activated lymphocytes.

CD26 is a lymphocyte marker that can anchor adenosine deaminase (ADA) on the T cell surface. We found that ADA is regulated by cytokines on the cell surface during T cell activation. By means of flow cytometry, immunofluorescence, and immunoblotting techniques, we found that interleukin (IL)-2 and IL-12 up-regulate ecto-ADA and CD26 expression. In clear contrast, IL-4 led to down-regulation of lymphocyte surface ADA without modifying the level of CD26. Moreover, neither circulating ADA transcription nor mRNA translation was regulated by cytokines. These results, along with absence of total-ADA modulation, the variable amount of ADA found in purified plasma membranes, and the different effect of Brefeldin A on the surface presence of ADA and CD26 indicated that cytokines regulate the translocation of ADA towards the cell surface through a mechanism not involving CD26. Ecto-ADA protected activated lymphocytes from the toxic effects of extracellular adenosine. Therefore, this cell surface ADA control might constitute part of the fine immunoregulatory mechanism of adenosine-mediated signaling through purinergic receptors in leukocytes.

Adenosine Deaminase↗

PubMed2000

Dopamine D1 and adenosine A1 receptors form functionally interacting heteromeric complexes.

The possible molecular basis for the previously described antagonistic interactions between adenosine A(1) receptors (A(1)R) and dopamine D(1) receptors (D(1)R) in the brain have been studied in mouse fibroblast Ltk(-) cells cotransfected with human A(1)R and D(1)R cDNAs or with human A(1)R and dopamine D(2) receptor (long-form) (D(2)R) cDNAs and in cortical neurons in culture. A(1)R and D(1)R, but not A(1)R and D(2)R, were found to coimmunoprecipitate in cotransfected fibroblasts. This selective A(1)R/D(1)R heteromerization disappeared after pretreatment with the D(1)R agonist, but not after combined pretreatment with D(1)R and A(1)R agonists. A high degree of A(1)R and D(1)R colocalization, demonstrated in double immunofluorescence experiments with confocal laser microscopy, was found in both cotransfected fibroblast cells and cortical neurons in culture. On the other hand, a low degree of A(1)R and D(2)R colocalization was observed in cotransfected fibroblasts. Pretreatment with the A(1)R agonist caused coclustering (coaggregation) of A(1)R and D(1)R, which was blocked by combined pretreatment with the D(1)R and A(1)R agonists in both fibroblast cells and in cortical neurons in culture. Combined pretreatment with D(1)R and A(1)R agonists, but not with either one alone, substantially reduced the D(1)R agonist-induced accumulation of cAMP. The A(1)R/D(1)R heteromerization may be one molecular basis for the demonstrated antagonistic modulation of A(1)R of D(1)R receptor signaling in the brain. The persistence of A(1)R/D(1)R heteromerization seems to be essential for the blockade of A(1)R agonist-induced A(1)R/D(1)R coclustering and for the desensitization of the D(1)R agonist-induced cAMP accumulation seen on combined pretreatment with D(1)R and A(1)R agonists, which indicates a potential role of A(1)R/D(1)R heteromers also in desensitization mechanisms and receptor trafficking.

2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-ben↗

PubMed2000

Evidence for adenosine/dopamine receptor interactions: indications for heteromerization.

Evidence has been obtained for adenosine/dopamine interactions in the central nervous system. There exists an anatomical basis for the existence of functional interactions between adenosine A(1)R and dopamine D(1)R and between adenosine A(2A) and dopamine D(2) receptors in the same neurons. Selective A(1)R agonists affect negatively the high affinity binding of D(1) receptors. Activation of A(2A) receptors leads to a decrease in receptor affinity for dopamine agonists acting on D(2) receptors, specially of the high-affinity state. These interactions have been reproduced in cell lines and found to be of functional significance. Adenosine/dopamine interactions at the behavioral level probably reflect those found at the level of dopamine receptor binding and transduction. All these findings suggest receptor subtype-specific interactions between adenosine and dopamine receptors that may be achieved by molecular interactions (e.g., receptor heterodimerization). At the molecular level adenosine receptors can serve as a model for homomeric and heteromeric protein-protein interactions. A1R forms homodimers in membranes and also form high-order molecular structures containing also heterotrimeric G-proteins and adenosine deaminase. The occurrence of clustering also clearly suggests that G-protein- coupled receptors form high-order molecular structures, in which multimers of the receptors and probably other interacting proteins form functional complexes. In view of the occurrence of homodimers of adenosine and of dopamine receptors it is speculated that heterodimers between these receptors belonging to two different families of G-protein-coupled receptors can be formed. Evidence that A1/D1 can form heterodimers in cotransfected cells and in primary cultures of neurons has in fact been obtained. In the central nervous system direct and indirect receptor-receptor interactions via adaptor proteins participate in neurotransmission and neuromodulation and, for example, in the establishment of high neural functions such as learning and memory.

Animals↗

PubMed2000

Adenosine A(1) receptor in cultured neurons from rat cerebral cortex: colocalization with adenosine deaminase.

Adenosine A(1) receptors (A(1)Rs) have been characterized in primary cultures of neurons from cerebral cortex. The specific adenosine A(1) antagonist 8-cyclopentyl-1,3-[(3)H]dipropylxanthine bound to both membranes and intact cells. When saturation experiments were performed in membranes, a K(D) value of 0.76 nM and a B(max) of 57 fmol/mg of protein were obtained. Competition assays revealed a pharmacological profile characteristic of A(1)Rs. The presence of this receptor was further confirmed by RT-PCR analysis. The expression of the receptor showed no significant changes during the period of culture studied, up to 12 days in vitro. A(1)R agonist inhibited forskolin-stimulated adenylyl cyclase, showing the functional coupling of these receptors with the effector. alphaG(i1, 2) protein level, detected by immunoblot, presented an increase during the period of culture. This increase correlated with an increase in the mRNA level of alphaG(i1) but not alphaG(i2). By immunochemical assays, it is shown that these receptors are expressed in both the neuronal cell body and the proximal dendrites. Colocalization of A(1)Rs with microtubule-associated protein 2 and cell surface adenosine deaminase was shown by confocal microscopy. The high degree of colocalization observed between A(1)Rs and ectoadenosine deaminase in neurons could suggest an important role of the enzyme in adenosine-mediated neuromodulation.

Adenosine Deaminase↗

PubMed2000

The heat shock cognate protein hsc73 assembles with A(1) adenosine receptors to form functional modules in the cell membrane.

A(1) adenosine receptors (A(1)Rs) are G protein-coupled heptaspanning receptors that interact at the outer face of the plasma membrane with cell surface ecto-adenosine deaminase (ecto-ADA). By affinity chromatography the heat shock cognate protein hsc73 was identified as a cytosolic component able to interact with the third intracellular loop of the receptor. As demonstrated by surface plasmon resonance, purified A(1)Rs interact specifically with hsc73 with a dissociation constant in the nanomolar range (0.5 +/- 0.1 nM). The interaction between hsc73 and A(1)R led to a marked reduction in the binding of the ligands and prevented activation of G proteins, as deduced from (35)S-labeled guanosine-5'-O-(3-thio)triphosphate binding assays. Interestingly this effect was stronger than that exerted by guanine nucleotide analogs, which uncouple receptors from G proteins, and was completely prevented by ADA. As assessed by immunoprecipitation a high percentage of A(1)Rs in cell lysates are coupled to hsc73. A relatively high level of colocalization between A(1)R and hsc73 was detected in DDT(1)MF-2 cells by means of confocal microscopy, and no similar results were obtained for other G protein-coupled receptors. Colocalization between hsc73 and A(1)R was detected in specific regions of rat cerebellum and in the body of cortical neurons but not in dendrites or synapses. Remarkably, agonist-induced receptor internalization leads to the endocytosis of A(1)Rs by two qualitatively different vesicle types, one in which A(1)R and hsc73 colocalize and another in which hsc73 is absent. These results open the interesting possibility that signaling via G protein-coupled receptors may be regulated by heat shock proteins.

Adenosine Deaminase↗

PubMed2000

Immunolocalization of A1 adenosine receptors in mammalian spermatozoa.

The presence of A1 adenosine receptors (A1AR) in mammalian spermatozoa was previously demonstrated by radiochemical and immunochemical detection. This study was performed to investigate the cellular location of the A1AR to determine whether these receptors were somehow connected with ecto-adenosine deaminase and to evaluate their function in calcium uptake. By immunofluorescence staining we showed that in mammalian spermatozoa A1AR were constantly localized in the acrosomal region. This finding was confirmed by immunogold detection. Confocal analyses with anti-A1 and anti-ADA antibodies showed a high degree of co-localization. Calcium loading assay showed that this association was functional and affected calcium accumulation in mammalian spermatozoa. Therefore, we concluded that the acrosomal localization of A1AR was a constant feature in mammalian sperm. Moreover, these A1 receptors were functionally coupled to ecto-ADA and were able to modulate calcium uptake into an IP3-gated store.(J Histochem Cytochem 48:1163-1171, 2000)

Adenosine Deaminase↗

PubMed1999

IFN-gamma up-regulates the A2B adenosine receptor expression in macrophages: a mechanism of macrophage deactivation.

Adenosine is a potent endogenous anti-inflammatory agent released by cells in metabolically unfavorable conditions, such as hypoxia or ischemia. Adenosine modulates different functional activities in macrophages. Some of these activities are believed to be induced through the uptake of adenosine into the macrophages, while others are due to the interaction with specific cell surface receptors. In murine bone marrow-derived macrophages, the use of different radioligands for adenosine receptors suggests the presence of A2B and A3 adenosine receptor subtypes. The presence of A2B receptors was confirmed by flow cytometry using specific Abs. The A2B receptor is functional in murine macrophages, as indicated by the fact that agonists of A2B receptors, but not agonists for A1, A2A, or A3, lead to an increase in cAMP levels. IFN-gamma up-regulates the surface protein and gene expression of the A2B adenosine receptor by induction of de novo synthesis. The up-regulation of A2B receptors correlates with an increase in cAMP production in macrophages treated with adenosine receptor agonist. The stimulation of A2B receptors by adenosine or its analogues inhibits the IFN-gamma-induced expression of MHC class II genes and also the IFN-gamma-induced expression of nitric oxide synthase and of proinflammatory cytokines. Therefore, the up-regulation of the A2B adenosine receptor expression induced by IFN-gamma could be a feedback mechanism for macrophage deactivation.

Adenosine↗

PubMed1999

Epidermal growth factor (EGF)-induced up-regulation and agonist- and antagonist-induced desensitization and internalization of A1 adenosine receptors in a pituitary-derived cell line.

This report concerns the study of homologous and heterologous regulation of cell surface A1 adenosine receptors (A1R) in a pituitary-derived cell line. This has been possible by the use of the recently developed anti-A1R antibodies in immunocytochemical assays. Functional desensitization and internalization of A1R in GH4 cells occurred after treatment with agonist but also with antagonist. Epidermal growth factor (EGF) treatment led to the up-regulation of cell surface A1R in GH4 cells. Confocal analysis evidenced an EGF-induced increase of A1R present in intracellular clathrin-coated vesicles. The up-regulation was blocked by actinomycin D thus suggesting the involvement of protein synthesis in the effect induced by the growth factor. These results constitute the first example of adenosine receptor regulation by EGF and one of the few examples of antagonist-induced desensitization and internalization among G-protein-coupled receptors.

Adenosine↗

PubMed1999

CD26 and adenosine deaminase interaction: its role in the fusion between horse membrane vesicles and spermatozoa.

Membrane vesicles of horse seminal plasma present at their surface a highly specific serine-type protease, dipeptidyl peptidase IV/CD26, a surface antigen known to characterize human prostasomes. Horse sperm cells expressed at their surface A(1) adenosine receptors (A(1)AR) and ecto-adenosine deaminase (ecto-ADA), both detected by immunoblot analysis, whereas CD26 was visualized at the equatorial segment by immunofluorescence microscopy. In addition to CD26, horse membrane vesicles showed ecto-ADA. The fusion process between horse sperm cells and vesicles was evidenced by confocal microscopy, which showed the localization of CD26 at the postacrosomal region and at the midpiece of the spermatozoa after incubation with vesicles. Moreover, a similar localization of CD26 and ecto-ADA on the spermatozoa was evidenced after fusion. Our results suggest that the interaction CD26/ecto-ADA might be responsible for fusion. Since A(1)ARs are said to be second receptors for ecto-ADA to form ecto-ADA/A(1)AR complexes, and since horse spermatozoa have A(1)ARs at their surface, the interaction CD26/ecto-ADA/A(1)AR during the fusion process cannot be ruled out.

Adenosine Deaminase↗