Tuberoinfundibular peptide of 39 residues (TIP39): molecular structure and activity for parathyroid hormone 2 receptor

Abstract

The neuropeptide TIP39 was recently purified from bovine hypothalamus based on the ability of the peptide to activate the parathyroid hormone 2 receptor (PTH2R) (Usdin et al. Nat. Neurosci. 2 (1999) 941). PTH2R is abundantly expressed in the nervous system, and its expression pattern suggests that it may play a role in modulation of pituitary function and in nociception. Towards understanding the physiological role of TIP39 and PTH2R, we cloned human, mouse and rat TIP39 gene. Our results revealed that: (1) the mature peptide is processed from a precursor; (2) TIP39 peptide is highly conserved among species; and (3) TIP39 from all species activates adenylyl cyclase and elevates intracellular calcium levels through PTH2R. We also defined and compared the structure–activity relationship of TIP39 on both activation of adenylyl cyclase and calcium mobilization pathways through PTH2R, finding common and differential determinants of TIP39 that are required for these pathways. Furthermore, we observed that TIP39 elevates intracellular calcium levels in primary dorsal root ganglion neurons whereas the peptide inactive on PTH2R do not, suggesting that TIP39 may activate these neurons important for nociception in vivo through PTH2R-dependent mechanisms.

Introduction

The parathyroid hormone (PTH) receptor 2 (PTH2R) was identified based on its homology to PTH/PTHrP receptor (PTHR) and was found to be abundantly expressed in the nervous system (Usdin et al., 1995, Usdin et al., 1999, Usdin et al., 2000, Usdin, 2000, Wang et al., 2000, Hoare and Usdin, 2001). Both PTHR and PTH2R are members of the type II G protein-coupled receptor superfamily that includes the receptors for glucagon, glucagon-like peptide-1, vasoactive intestinal protein, CRF, secretin, and calcitonin (Abou-Samra et al., 1992, Segre, 1996). PTH is known as a principal modulator of mineral ion homeostasis (Potts et al., 1995). This peptide acts on PTHR to elevate blood calcium levels in bone and kidney tissues. PTH-related protein (PTHrP) can also activate the PTHR, and is believed to be involved in the maintenance of numerous tissues and to be an important developmental regulator (Grill et al., 1998). Both PTH and PTHrP stimulate cAMP accumulation and PLC/PKC pathways through activation of PTHR (Juppner et al., 1991, Abou-Samra et al., 1992, Bringhurst et al., 1993, Schneider et al., 1993, McCuaig et al., 1994, Pines et al., 1994, Iida-Klein et al., 1995, Iida-Klein et al., 1997, Segre, 1996, Smith et al., 1996, Potts and Juppner, 1997, Gardella and Juppner, 2001, Hoare and Usdin, 2001). PTH peptide and the more recently discovered neuropeptide TIP39, but not PTHrP, activate PTH2R in vitro (Abou-Samra et al., 1992, Usdin et al., 1995, Gardella and Juppner, 2001, Hoare and Usdin, 2001). Despite the in vitro activity of PTH on PTH2R, PTH is unlikely to be an endogenous ligand for PTH2R in the central nervous system (CNS) because: PTH is not found in the CNS; whereas, PTH2R is abundantly expressed in the CNS (Usdin et al., 1995, Usdin et al., 1999, Usdin et al., 2000, Wang et al., 2000, Hoare and Usdin, 2001); and PTH has little activity in stimulating cAMP accumulation and elevating intracellular calcium on rat PTH2R (Usdin et al., 1999, Goold et al., 2001). Recently, a distinct peptide, TIP39, was purified from bovine brain based on its activity on PTH2R in vitro (Usdin et al., 1999). Bovine TIP39 appears to be distantly related to PTH and PTHrP. PTH(1-34) and PTHrP(1-36) show sequence similarity only in the N-terminal 13 amino acids, eight of which are identical. Bovine TIP39 contains four of the eight residues shared by mammalian PTH and PTHrP. When similar residues are considered, the sequence similarity between the three ligands approaches 50% (Hoare and Usdin, 2001). The secondary structures of PTH and TIP39 also appear to be similar, consisting of two α-helices (Piserchio et al., 2000). Bovine TIP39 potently activates the human and rat PTH2R but has little or no effect on PTHR (Usdin et al., 1999).

Clues about the biological function of the PTH2R and TIP39 came from studies of cellular distribution of the receptor in the nervous system (Usdin et al., 1999, Wang et al., 2000). PTH2R was observed to be expressed in the external zone of the median eminence, an anatomical region important for the regulation of pituitary hormone secretion. Dorsal root ganglion sensory neurons also express the PTH2R. Expression of PTH2R protein was also detected in the superficial layers in the dorsal horn of the spinal cord which are involved in nociception (Usdin et al., 1999, Wang et al., 2000, Dobolyi et al., 2002). Although the physiological function of TIP39 and the PTH2R is unknown, the anatomical distribution of the PTH2R suggests that TIP39 might be involved in the modulation of processes that range from pituitary hormone release to nociception. Very recently, it was demonstrated that TIP39 is expressed in regions in the brain that innervate the spinal cord, consistent with TIP39 being an endogenous ligand for PTH2R (Dobolyi et al., 2002, John et al., 2002). In animal tests of nociception, TIP39 peptide was shown to strongly potentiate nociception (Dobolyi et al., 2002). In addition, the TIP39 genomic structure has been revealed based on the genomic sequence in the database (Dobolyi et al., 2002, John et al., 2002). The predicted sequences of mouse TIP39 and human TIP39 are highly homologous, and the processing of the peptide precursor was also predicted based on the genomic sequences (John et al., 2002).

In this paper, we describe the cloning of the cDNA clones for human, mouse and rat TIP39, and the processing of the peptide from the precursors. We used an approach that directly cloned the cDNA encoding TIP39 from human, mouse and rat nervous systems to provide information on the molecular structure of the cDNA and the processing of TIP39 peptide precursor. We also characterized the activity of the peptides from different species on the PTH2R. We found that in addition to activation of adenylyl cyclase pathway, TIP39 elevates intracellular calcium levels in cells that express PTH2R through calcium mobilization from intracellular stores. We also investigated the structure–activity relationship for ligand activation of PTH2R in both the activation of adenylyl cyclase and in the elevation of intracellular calcium. Furthermore, in order to examine whether TIP39 may indeed play a role in nociception by activation of sensory neurons through similar pathways as in transfected cells, we examined intracellular calcium mobilization by TIP39 in dorsal root ganglion neurons.