Glp-Asn-Pro-D-Tyr-D-TrpNH(2) is a novel synthetic peptide that mimics and amplifies central actions of thyrotropin-releasing hormone (TRH) in rat without releasing TSH. The aim of this study was to compare the binding properties of this pentapeptide and its all-L counterpart (Glp-Asn-Pro-Tyr-TrpNH(2)) to TRH receptors in native rat brain tissue and cells expressing the two TRH receptor subtypes identified in rat to date, namely TRHR1 and TRHR2. Radioligand binding studies were carried out using [(3)H][3-Me-His(2)]TRH to label receptors in hippocampal, cortical and pituitary tissue, GH4 pituitary cells, as well as CHO cells expressing TRHR1 and/or TRHR2. In situ hybridization studies suggest that cortex expresses primarily TRHR2 mRNA, hippocampus primarily TRHR1 mRNA and pituitary exclusively TRHR1 mRNA. Competition experiments showed [3-Me-His(2)]TRH potently displaced [(3)H][3-Me-His(2)]TRH binding from all tissues/cells investigated. Glp-Asn-Pro-D-Tyr-D-TrpNH(2) in concentrations up to 10(-5)M did not displace [(3)H][3-Me-His(2)]TRH binding to membranes derived from GH4 cells or CHO-TRHR1 cells, consistent with its lack of binding to pituitary membranes and TSH-releasing activity. Similar results were obtained for the corresponding all-L peptide. In contrast, both pentapeptides displaced binding from rat hippocampal membranes (pIC(50) Glp-Asn-Pro-D-Tyr-D-TrpNH(2): 7.7+/-0.2; pIC(50) Glp-Asn-Pro-Tyr-TrpNH(2): 6.6+/-0.2), analogous to cortical membranes (pIC(50) Glp-Asn-Pro-D-Tyr-D-TrpNH(2): 7.8+/-0.2; pIC(50) Glp-Asn-Pro-Tyr-TrpNH(2): 6.6+/-0.2). Neither peptide, however, displaced [(3)H][3-Me-His(2)]TRH binding to CHO-TRHR2. Thus, this study reveals for the first time significant differences in the binding properties of native and heterologously expressed TRH receptors. Also, the results raise the possibility that Glp-Asn-Pro-D-Tyr-D-TrpNH(2) is not displacing [(3)H][3-Me-His(2)]TRH from a known TRH receptor in rat cortex, but rather a hitherto unidentified TRH receptor.