RESUMEN
Herein, we have used bioinformatics tools to predict five clusters defining ligand-binding sites on the extracellular domain of human CD300b receptor, presumably involved in the formation of both homodimers and heterodimers with other CD300 family members. Site-directed mutagenesis revealed residues glutamic acid 28 and glutamine 29 in cluster 5 to be necessary for the formation of CD300b complexes. Surprisingly, the disruption of cluster 2 and 4 reconstituted the binding capability lost by the mutation of residues glutamic acid 28 to alanine, glutamine 29 to alanine (E28A-Q29G). We identified a missense mutation arginine 33 to glutamine (R33Q) in CD300f by direct sequencing of exon 2 in peripheral blood samples from 50 patients with multiple sclerosis (MS). Levels of expression of CD300f were almost undetectable on monocytes from the patient bearing the R33Q mutation compared with healthy individuals. Whereas R33Q mutation had no effect in the formation of CD300f complexes, the inhibition of protein synthesis with cycloheximide indicated that CD300f R33Q is less stable than native CD300f. Finally, we report that the levels of expression of CD300f on the surface of classical and intermediate monocytes from MS patients are significantly lower when compared to the same cell populations in healthy individuals.
Asunto(s)
Esclerosis Múltiple/patología , Receptores Inmunológicos/metabolismo , Adulto , Secuencia de Aminoácidos , Animales , Sitios de Unión , Células COS , Estudios de Casos y Controles , Chlorocebus aethiops , Cicloheximida/metabolismo , Femenino , Expresión Génica , Humanos , Ligandos , Masculino , Monocitos/citología , Monocitos/metabolismo , Esclerosis Múltiple/genética , Mutagénesis Sitio-Dirigida , Polimorfismo de Nucleótido Simple , Estructura Terciaria de Proteína , Receptores Inmunológicos/química , Receptores Inmunológicos/genéticaRESUMEN
Herein we present the cloning and molecular characterization of CD300d, a member of the human CD300 family of immune receptors. CD300d cDNA was cloned from RNA obtained from human peripheral blood mononuclear cells, and RT-PCR revealed the gene to be expressed in cells of myeloid lineage. The cloned cDNA encoded for a type I protein with a single extracellular Ig V-type domain and a predicted molecular mass of 21.5 kDa. The short cytoplasmic tail is lacking in any known signaling motif, but there is a negatively charged residue (glutamic acid) within the transmembrane domain. CD300d forms complexes with the CD300 family members, with the exception of CD300c. Contrary to other activating members of the CD300 family of receptors, surface expression of CD300d in COS-7-transfected cells required the presence of an immunoreceptor tyrosine-based activating motif-bearing adaptor (FcεRγ). Accordingly, we found that CD300d was able to recruit FcεRγ. Unexpectedly, we could not detect CD300d on the surface of cells expressing FcεRγ, suggesting the existence of unknown mechanisms regulating the trafficking of this molecule. The presence of other CD300 molecules also did not modify the intracellular expression of CD300d. In fact, the presence of CD300d decreased the levels of surface expression of CD300f but not CD300c. Our data suggest that the function of CD300d would be related to the regulation of the expression of other CD300 molecules and the composition of CD300 complexes on the cell surface.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Leucocitos Mononucleares/metabolismo , Complejos Multiproteicos/metabolismo , Receptores Inmunológicos/biosíntesis , Receptores Inmunológicos/genética , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Células COS , Chlorocebus aethiops , Clonación Molecular , ADN Complementario/genética , Células HeLa , Humanos , Leucocitos Mononucleares/citología , Datos de Secuencia Molecular , Complejos Multiproteicos/genética , Estructura Terciaria de Proteína , Transporte de Proteínas , Receptores de IgE/genética , Receptores de IgE/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
It is well known that cell surface immune receptors play a critical role in regulating immune and inflammatory processes in the central nervous system (CNS). We have analyzed the function of cluster of differentiation (CD)300f immunoreceptor in a model of excitotoxic rat brain damage. First, to explore the presence of endogenous ligand(s) for this receptor we used a human CD300f-Ig soluble protein and confocal microscopy, showing specific staining mainly in CNS white matter and on the surface of oligodendrocytes and certain astrocytes. Next, we demonstrated in a model of in vivo rat brain excitotoxic damage that the overexpression of human CD300f induced a significant reduction in the lesion volume. To validate these results, we cloned the rat ortholog of CD300f protein (rCD300f). The overexpression of rCD300f receptor had a comparable neuroprotective effect after the acute brain injury and a similar CNS staining pattern when stained with the rCD300f-Ig soluble protein. Interestingly, when we analyzed the expression pattern of rCD300f in brain cells by quantitative polymerase chain reaction and immunohistochemistry, we detected the expression of CD300f as expected in microglial cells, but also in oligodendrocytes and neurons. These data suggest that the neuroprotective role of CD300f would be the result of a complex network of cell interactions.
Asunto(s)
Lesiones Encefálicas/metabolismo , Encéfalo/metabolismo , Microglía/metabolismo , Neuronas/metabolismo , Oligodendroglía/metabolismo , Receptores Inmunológicos/metabolismo , Animales , Astrocitos/metabolismo , Astrocitos/patología , Encéfalo/patología , Lesiones Encefálicas/inducido químicamente , Lesiones Encefálicas/patología , Células Cultivadas , Humanos , Microglía/patología , Neuronas/patología , Oligodendroglía/patología , Ratas , Receptores Inmunológicos/genéticaRESUMEN
The CD300 family of myeloid immunoglobulin receptors includes activating (CD300b, CD300e) and inhibitory members (CD300a, CD300f), as well as molecules of uncertain function presenting a negative charge within their transmembrane domain (CD300c, CD300d). In this paper, we establish that CD300c is a functional immune receptor able to deliver activating signals upon ligation in RBL-2H3 mast cells. CD300c signaling is partially mediated by a direct association with the immune receptor tyrosine-based activation motif-bearing adaptor FcεRγ. The existence of complementary transmembrane-charged residues in certain CD300 receptors suggested the formation of heterodimers within this family. Indeed, we proved the interaction between CD300b and CD300c in transfected COS-7 cells and demonstrated that it has important functional consequences. Unexpectedly, dimmer formation was dependent on the immunoglobulin domains rather than the charged transmembrane residues. Concordantly, all CD300 members were found to interact with each other, even with themselves, forming both homo- and heterodimers. We found that the combination of CD300 receptors in a complex differentially modulates the signaling outcome, strongly suggesting a new mechanism by which CD300 complexes could regulate the activation of myeloid cells upon interaction with their natural ligands.