Expression analysis of the thyrotropin-releasing hormone receptor (TRHR) in the immune system using agonist anti-TRHR monoclonal antibodies.
Monoclonal anti-rat thyrotropin-releasing hormone (TRH) receptor (TRHR)-specific antibodies (mAb) were generated by immunization with synthetic peptides of rat TRHR partial amino acid sequences; one (TRHR01) was directed against a sequence (84-98) in the extracellular portion of the rat TRHR reported to be constant among different species, including man, and the second (TRHR02) recognizes the C-terminal region sequence 399-412. In lysates from GH4C1 cells, a clonal rat pituitary cell line, both mAb recognize the TRHR in Western blot analysis, and TRHR02 immunoprecipitates the TRHR. Incubation of GH4C1 cells with the mAb causes a fluorescence shift in fluorescence-activated cell sorting analysis. The cells were stained specifically by both mAb using immunocytochemical techniques. Furthermore, TRHR01 is agonistic in its ability to trigger Ca2+ flux, and desensitizes the TRH receptor. We tested for TRHR in several rat organs and found expression in lymphoid tissues. TRHR01 recognizes the human TRHR, and analysis of human peripheral blood lymphocyte and tonsil-derived leukocyte populations showed receptor expression in non-activated and phytohemagglutinin-activated T and B cells.
Mellado
M
M
Department of Immunology and Oncology, Centro Nacional de Biotecnología (CSIC), Madrid, Spain. mmellado@cnb.uam.es
Fernández-Agulló
T
T
Rodríguez-Frade
J M
JM
San Frutos
M G
MG
de la Peña
P
P
Martínez-A
C
C
Montoya
E
E
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D002460:Cell Line; D005434:Flow Cytometry; D006801:Humans; D007107:Immune System / Q000378:metabolism*; D008213:Lymphocyte Activation; D008221:Lymphoid Tissue / Q000276:immunology / Q000378:metabolism*; D051379:Mice; D009928:Organ Specificity; D051381:Rats; D018025:Receptors, Thyrotropin-Releasing Hormone / Q000819:agonists / Q000276:immunology / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D018025:Receptors, Thyrotropin-Releasing Hormone