Production and characterization of polyclonal anti-idiotypic anti-TRH antibodies: application to the study of pituitary TRH receptor.
cDNA encoding the thyrotropin-releasing hormone receptor (TRH-R) was recently cloned in rat pituitary prolactin cells and in mouse thyrotropes. The molecular weights of the protein sequences obtained are 46.6 and 44.5 kD. However, TRH-R has not yet been purified to homogeneity and specific anti-TRH-R antibody could not yet be obtained by classical biochemical methods. We thus attempted to obtain antibodies specific for TRH-R using an anti-idiotypic approach. Rabbits of the same allotype were immunized using Igs (Ab1) extracted from rabbit polyclonal anti-TRH immune serum. Anti-idiotypic rabbit polyclonal anti-anti-TRH antibodies (Ab2) were obtained, as shown by their ability to inhibit the formation of TRH-anti-TRH complexes in a radioimmunoassay system. One of them, the polyclonal Ab2 R38/B12, was tested for its ability to recognize the TRH-R in rat pituitary, tumor-derived, GH3/B6 prolactin-secreting cells. Immunoreactive material was immunocytochemically detected in fixed and saponin-permeabilized GH3/B6 cells. The immunostaining was localized at the plasma membrane and on intracellular structures. It was not observed using non-anti-TRH Ab2 and was abolished in the presence of excess TRH. Furthermore, binding of [125I]R38/B12 on fixed and saponin-permeabilized GH3/B6 cells was partially inhibited by excess TRH. By immunoblot analyses of Triton X-114 cell extracts performed under reducing or nonreducing conditions, the polyclonal R38/B12 Igs revealed two main protein species of approximately 98 and approximately 76 kD as well as several proteins < or = 46 kD. In the presence of excess TRH, the approximately 98- and approximately 42-kD bands were abolished, whereas the intensity of the other bands was faintly attenuated only. The approximately 98-kD protein was also revealed in a two-dimensional PAGE analysis. Nevertheless, the effects of R38/B12 Igs on [3H]TRH binding by GH3/B6 cells and on basal or TRH-induced prolactin secretion were not markedly different from those elicited by control Ab2. These data suggest that we have characterized Ab2 antibodies which recognize a molecular entity that might be related to the TRH-R in GH3B6 cells.
Grouselle
D
D
Groupe de Biologie de la Cellule Neuroendocrine, Collĕge de France, Paris.
Roland
J
J
Rousselet
A
A
Denoulet
P
P
Cazenave
P A
PA
Tixier-Vidal
A
A
Gourdji
D
D
D000818:Animals; D000888:Antibodies, Anti-Idiotypic / Q000096:biosynthesis* / Q000276:immunology*; D000918:Antibody Specificity; D005455:Fluorescent Antibody Technique; D013006:Growth Hormone / Q000378:metabolism; D015151:Immunoblotting; D010902:Pituitary Gland / Q000737:chemistry*; D010911:Pituitary Neoplasms / Q000737:chemistry / Q000378:metabolism; D011388:Prolactin / Q000378:metabolism; D051381:Rats; D018025:Receptors, Thyrotropin-Releasing Hormone / Q000032:analysis*; D012503:Saponins; D013973:Thyrotropin-Releasing Hormone / Q000276:immunology* / Q000378:metabolism; D014407:Tumor Cells, Cultured
D000888:Antibodies, Anti-Idiotypic; D018025:Receptors, Thyrotropin-Releasing Hormone; D012503:Saponins; D013973:Thyrotropin-Releasing Hormone; D011388:Prolactin; D013006:Growth Hormone