Production and characterization of an antiserum which recognizes the native receptor for thyrotropin-releasing hormone

Despite attempts in several laboratories, it has been difficult to prepare antiserum to the thyrotropin-releasing hormone receptor (TRHR). We have pre pared a polyclonal anti-rat TRHR antiserum. by immunization of rabbits with a synthetic peptide corresponding to the C-terminus of the TRHR. The speci ficity of the antiserum was assessed by enzyme-linked immunosorbent assay. The affinity-purified antibody recognized a major broad band at 50-60 kDa a nd a minor broad band at 100-120 kDa in Western blot analysis of membrane p roteins from TRHR-transfected, but not control, HEK293t cells. Binding to b oth bands was abolished by preincubation with the immunizing peptide but no t control peptide. The approach was repeated with rat pituitary F4C1 cells, which lack endogenous TRHRs; membranes from F4C1 cells transfected with TR HR cDNA, but not control cells, showed specific binding by Western blot. Us ing laser confocal microscopy, the TRHR was visualized on the plasma membra ne of transfected, but not control, F4C1 cells. Similar confocal. findings were observed in TRHR-transfected HEK293t cells. Within 5 min after TRH add ition, the TRHR signal translocated from the plasma membrane to the cytopla sm of F4C1 cells transfected with TRHR cDNA. Ten minutes after TRH addition , the TRHR signal formed aggregates in the cytoplasm. Thirty minutes after TRH treatment, both cytoplasmic and plasma membrane localizations were obse rved, suggesting recycling of some TRHRs back to the plasma membrane. These observations are consistent with our previous findings using an epitope-ta gged TRHR. In conclusion, we have prepared an antiserum that recognizes the native TRHR by Western blot analysis and confocal microscopy. (C) 2001 Aca demic Press.