Development of a rat parathyroid hormone 2 receptor antagonist

Abstract

The parathyroid hormone 2 (PTH2) receptor is a Family B G-protein coupled receptor most highly expressed within the brain. Current evidence suggests that tuberoinfundibular peptide of 39 residues (TIP39) is the PTH2 receptor's endogenous ligand. To facilitate investigation of the physiological function of the PTH2 receptor/TIP39 system we have developed a novel PTH2 receptor antagonist, by changing several residues within the amino terminal domain of TIP39. Histidine 4 , tyrosine 5 , tryptophan 6 , histidine 7 -TIP39 binds the PTH2 receptor with high affinity, has over 30-fold selectivity for the rat PTH2 receptor over the rat PTH1 receptor and displays no detectable agonist activity. This ligand should be useful for in vivo investigation of PTH2 receptor function.

Thalamic neuropeptide mediating the effects of nursing on lactation and maternal motivation

“…Two lentiviral vectors were prepared: a HYWH-GFP virus was designed to express a secreted form of histidine 4 , tyrosine 5 , tryptophan 6 , histidine 7 -TIP39 (HYWH-TIP39) an antagonist of the PTH2 receptor (Kuo and Usdin, 2007), plus GFP that remained within infected cells and allowed their visualization, while a control virus expressed GFP only. To produce th HYWH-GFP lentiviral vector, we used a combination of oligonucleotide synthesis, PCR, and conventional subcloning techniques.…”

“…To produce th HYWH-GFP lentiviral vector, we used a combination of oligonucleotide synthesis, PCR, and conventional subcloning techniques. In the resulting construct, a strong mammalian promoter (EF-1α) drives expression of a fusion protein between the fibronectin leader sequence with signal peptide cleavage site and the HYWH-TIP39 sequence a PTH2 receptor antagonist (Kuo and Usdin, 2007). This is followed by an internal ribosome reentry site (IRES) and then enhanced green fluorescent protein (EGFP) sequence and a woodchuck hepatitis post-transcriptional regulatory element (WPRE) as described previously (Dimitrov et al, 2013).…”

Regulation of Hypothalamic Signaling by Tuberoinfundibular Peptide of 39 Residues Is Critical for the Response to Cold: A Novel Peptidergic Mechanism of Thermoregulation

“…On the day of the experiment, after one-hour acclimation to the procedure room, a 33-gauge internal injector with a 0.5 mm projection was inserted into the cannula and 0.1 µl of vehicle containing 0.1% bovine serum albumin (Equitech-Bio Inc., Kerrville, TX) in saline or 50, 100 and 200 pmol of TIP39 was delivered over 5 minutes. The lateral ventricle injections were performed in the same way with a dose of 500 pmol TIP39 or H 3 Y 4 W 5 H 6 -TIP39 (HYWH, a PTH2R antagonist (Kuo and Usdin, 2007) ) in 0.5 µl vehicle. The injectors were removed one minute after completion of the injection and the animal’s body temperature was monitored for 7 to 8 hours.…”

International Union of Basic and Clinical Pharmacology. XCIII. The Parathyroid Hormone Receptors—Family B G Protein–Coupled Receptors

“…In a search for novel ligands that would act as selective antagonists for the PTHR2, and thus ultimately might be useful in defining the biologic role of this receptor in vivo, Kuo and Usdin (2007) performed a sitedirected mutagenesis-based analysis of the TIP39 sequence, searching the mutants for absence of cAMP signaling activity on the PTHR2. The study yielded the TIP(1-39) analog having the sequence Leu-Ala-Asp-Asp at positions 4-7 replaced by the sequence His-Tyr-TrpHis.…”

“…The study yielded the TIP(1-39) analog having the sequence Leu-Ala-Asp-Asp at positions 4-7 replaced by the sequence His-Tyr-TrpHis. In cell-based assays, the new analog, called HYWH-TIP39, was shown to be 15-fold more effective for inhibiting agonist-induced signaling at the PTHR2 than at the PTHR1 (Kuo and Usdin, 2007). Thus, HYWH-TIP39, indeed shows the opposite receptor antagonist selectivity profile as TIP(7-39).…”