Elsevier

Peptides

Volume 23, Issue 9, September 2002, Pages 1607-1615
Peptides

Cloning and characterization of murine neuromedin U receptors

https://doi.org/10.1016/S0196-9781(02)00097-9Get rights and content

Abstract

Neuromedin U (NmU) is a neuropeptide involved in various physiological functions such as feeding behavior, muscle contractile activity, and regulation of intestinal ion transport. Recently, two human G protein-coupled receptors have been identified as NmU-specific receptors, NmU-R1 and NmU-R2, which share 55% amino acid identity. It is unclear however, which of the two receptors mediates responses to NmU observed in rodent models. Attempts to define the pharmacological profile of the two receptors are confounded by overlapping expression of the two receptors and a lack of subtype-selective compounds. In order to establish a basis to further our understanding of the function of these receptors, we cloned and characterized the mouse homologues of the two human NmU receptors. Mouse NmU-R1 and mouse NmU-R2 are 79 and 81% identical to their respective human homologues. Expression of NmU-R1 was mainly observed in testis, gastrointestinal (GI) tract, and immune system, while NmU-R2 was primarily expressed in brain tissues. Each mouse receptor was independently expressed in HEK293 cells and demonstrated a dose-dependent calcium flux in response to NmU-8, NmU-23 and NmU-25. In an attempt to identify a synthetic NmU peptide that would exhibit selectivity at one of the two receptors, we examined the functional activity of eight alanine-substituted NmU-8 peptides. These experiments demonstrated that alanine substitution at positions 5 and 7 affects the functional activity of the peptide at both receptors. The arginine residue at position 7 is required for NmU-8 activity at either receptor while alanine substitution at position 5 selectively affects the potency and the efficacy at mNmU-R1. These experiments validate the use of rodent models to characterize NmU function relative to humans and suggest that substitution at Arginine-5 of NmU-8 may provide a receptor selective peptide.

Introduction

Neuromedin U (NmU) is an amidated peptide that is generated by enzymatic cleavage at the C-terminal region of a 174 amino acid precursor [24]. The first NmU peptides identified (NmU-8 and NmU-25) were isolated from porcine spinal cord [28]. It was suggested that NmU-8 was most likely processed from NmU-25 since it represented the C-terminal region of NmU-25 and was located just after a typical processing signal Arg–Arg. In other species, such as rabbit [21], rat [9], frog [11], [38] and chicken [33] NmU-8 is not generated because the NmU peptides all lack the Arg–Arg cleavage site. Subsequently, NmU has been isolated from rat [8], [9], human [3], guinea pig [29], rabbit [21] and dog [32]. It has also been shown that the C-terminal heptapeptide core of NmU (Phe–Leu–Phe–Arg–Pro–Arg–Asn–NH2) constitutes a conserved domain and is sufficient for the biological activity of the peptide [16].

In mammalian species, NmU is widely distributed with a predominant expression in gastrointestinal (GI) tract and central nervous system [1], [2], [4]. In human tissue, mRNA encoding for NmU precursor is found in intestine, stomach, pituitary gland and bone marrow as well as in several brain regions including hypothalamus, locus coeruleus, thalamus, medulla, and substantia nigra [17], [35], [41]. Interestingly, NmU mRNA expression is also found in lymphoid cells such as dendritic cells, monocytes and B cells suggesting a potential role of NmU in the regulation of immune functions [17], [43]. The pattern of distribution of NmU mRNA in rat tissue is similar to that observed in human tissues with NmU mRNA present in the small intestine, rectum, colon as well as in the pituitary gland and the genitourinary tract [3], [9], [12]. Moderate expression of NmU mRNA has also been reported in rat central nervous system regions such as nucleus accumbens, hypothalamus, septum, and spinal cord [4], [10], [18].

The first biological activity attributed to NmU is its contractile activity on smooth muscle cells [5], [25], [28], [34]. It has been shown that the C-terminal asparaginamide structure and the C-terminal heptapeptide core of NmU are essential for its contractile activity [28], [37]. Further structure–activity studies have been done in isolated chicken crop smooth muscle preparations using NmU-8 analogs substituted with glycine [16], [23], [36]. However, no data are available about the contribution of each NmU-8 residue to the functional activity of the peptide at the cloned receptors. In addition to its well-characterized smooth muscle contractile activity, several other physiological functions have been attributed to NmU. NmU has been shown to regulate adrenocortical functions [26], [27], to increase systemic blood pressure in rats [14], [40] and to alter ion transport in the porcine jejunum [7]. Studies investigating the physiological role of centrally administered NmU in rats have demonstrated that NmU acts as a potent anorectic peptide [20]. Intracerebroventricular administration of NmU in rats induces a decrease in food intake and body weight, and an increase in energy expenditure [30]. The presence of NmU in the arcuate nucleus of the hypothalamus, as well as the downregulation of NmU mRNA expression upon fasting further supports this hypothesis [6], [20].

Two receptors have been identified for NmU: NmU-R1 and NmU-R2, both of which have been shown to mediate an increase in the intracellular Ca2+ level in response to NmU peptides [17], [20], [35], [39]. However, it is not clear which of the receptors mediates the different physiological functions induced by NmU. Studies of the distribution of the two human receptors (hNmU-Rs: hNmU-R1 and hNmU-R2) have shown that while the level of expression of NmU-R2 is higher in central nervous system tissues, hNmU-R1 mRNA is expressed mainly in peripheral tissues, with little or no expression in central nervous system tissues [12], [17], [40]. In particular, hNmU-R1 mRNA is expressed in pancreas, testis, stomach, small intestine, liver, kidney and urogenital system [17], [20], [35]. Expression of NmU-R1 is also observed in some immune cells and tissues [17], [20], [43]. The expression of NmU-R2 is found mainly in central nervous system tissues although NmU-R2 mRNA is also detected in testis, lung, kidney, and thyroid [20], [39].

While a considerable amount is known about the human NmU-Rs little is known about the mouse counterparts (mNmU-Rs: mNmU-R1 and mNmU-R2). Murine genetic models have become a widely used tool in the study of human receptor function, therefore it is important that mouse orthologues are cloned and characterized. We report here the cloning and characterization of the murine NmU receptors (mNmU-R1 and mNmU-R2). The results indicate that the mouse receptors are distributed in a manner similar to their human counterparts, and that they share similar functional profiles. In addition, we have defined the amino acid residues of the NmU peptide which are critical for activation of the two receptors. The results of this study provide a basis for the interpretation of data generated from genetic manipulation of the NmU receptors, and help define the structural requirements for NmU receptor activation.

Section snippets

Cloning and expression of mouse and human NmU receptors

A partial cDNA sequence corresponding to the first 700 bp of the mNmU-R2 coding sequence was determined using the rat NmU-R2 cDNA sequence (GenBank AF242875) [20]. This was subsequently used to design a 5′ gene-specific primer starting at the initiating codon (5′-ATG GGA AAA CTT GAA AAT GCT TCC TGG ATC CAC GA-3′). To determine the 3′-end of the coding sequence of mNmU-R2, a PCR amplification was performed using mouse spinal cord 5′-stretch lambda cDNA library Triplex (Clonetech) using the 5′

Mouse and human NmU-Rs amino acid sequence shared high percentage of identity

The cDNA encoding mNmU-R2 was isolated from mouse spinal cord by PCR. The ORF of mNmU-R2 is 1188 bp long, giving a predicted amino acid sequence of 396 residues (GenBank AY057384). Amino acid alignment of mouse NmU-R2 and its human homologue (GenBank AF242874) showed 81% identity (Fig. 1) with the greatest divergence in the amino-terminus. Both human and mNmU-R2 share core characteristics of the GPCR family including seven predicted alpha-helice transmembrane domains, the ERY variant of the DRY

Discussion

The physiological functions thus far attributed to NmU suggest that NmU-receptors represent potential therapeutic targets. However, further studies are needed to determine the role of each receptor in mediating the different functions induced by NmU. Because there are no selective receptor antagonists available, the determination of the respective functions of the two NmU receptors will likely depend on the use of mouse gene knockouts. In order to establish a basis to further our understanding

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