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Transmitters and Hormones

Molecular Studies of the
Mammalian Neuropeptide
Tachykinin System

S. Nakanishi

Institute for Immunology, Kyoto University Faculty of Medicine
Kyoto 606, Japan

Peptidergic neurons play an important role in controlling the specificity and complexity of interneuronal communications. To understand the function and regulation of peptidergic neurons, it is necessary to elucidate detailed cellular mechanisms of peptidergic neurons, including peptide production, receptor function, intracellular signaling, and modulation of ion channel. Our studies of molecular and genetic aspects of the mammalian neuropeptide tachykinin system address some of these mechanisms.

Preprotachykinin Genes

The mammalian tachykinin system consists of three distinct peptides – substance P, substance K, and neuromedin K (reviewed in Nakanishi 1986, 1987). We have determined the polypeptide sequences, mRNA sequences, and gene organizations of the tachykinin precursors (preprotachykinins) by molecular cloning and sequence analyses of their cDNAs and genomic DNAS (Nawa et al. 1983, 1984; Kawaguchi et al. 1986; Kotani et al. 1986). Figure 1 schematically illustrates the structures and expressions of the preprotachykinin genes.

The three mammalian tachykinins are derived from two different but related genes. The preprotachykinin A gene encodes the precursors for substance P and substance K, while the preprotachykinin B gene specifies the precursor for neuromedin K. The two genes exhibit a marked structural

similarity in terms of exon-intron arrangements, suggesting that they evolved from a common ancestor gene.

The major expression sites of the two genes, however, differ markedly from each other: the preprotachykinin A mRNA is expressed mainly in the trigeminal ganglion and the striatum, whereas the preprotachykinin B mRNA is produced around the hypothalamus and in the intestines. In both genes, alternative RNA processing events are tightly associated with the gene expressions and result in the formation of multiple species of mRNAs. In the expression of the preprotachykinin A gene, three different mRNAs are generated from the single gene as a result of alternative RNA splicing. Furthermore, substance K is specified by a discrete genomic segment, and the generation of substance K is regulated in a tissue-specific manner through the inclusion or exclusion of the substance K-coding exon by different RNA splicing.

The preprotachykinin B gene also encodes two mRNAs and, in this case, the two mRNAs differ at the 5' extremity of the 5'-untranslated region as a result of differential usage of two different promoters. The mammalian tachykinin neuropeptides thus exhibit their diversity by effectively utilizing cellular mechanisms characteristic of eukaryotic cells, including gene duplication, differential expression of duplicated genes, and alternative RNA processing.

Tachykinin Receptors

Since the presence of a new tachykinin, substance K, was revealed by the nucleotide sequence analysis of the cloned preprotachykinin A cDNA, we chemically synthesized substance K and examined its biological activities in various pharmacological tests (Nawa et al. 1984). The result indicated that substance K possesses biological activities characteristic of the tachykinin peptides but markedly differs from substance P in its biological potencies. Thus, substance K represents a second type of mammalian tachykinin, which may differ from substance P in its physiological roles.

The differing patterns of biological activity between substance P and substance K also suggested the presence of multiple forms of tachykinin receptors in mammalian tissues. Several laboratories, by developing a ligandbinding assay, suggested the existence of at least three distinct receptors, each specific for one of the three mammalian tachykinins (reviewed in Quirion 1985). However, the tachykinin receptors represent minor cellular components and are tightly embedded in plasma membrane. Therefore, the nature of the tachykinin receptors, as with other neuropeptide receptors, remained to be clarified.

To circumvent this problem, we attempted a new approach to characterize these proteins by developing a Xenopus oocyte expression system combined

with electrophysiological measurements (Harada et al. 1987). The injection of an appropriate exogenous mRNA into a Xenopus oocyte induces a functional, foreign receptor-channel complex in the oocyte membrane, and the expression of the receptor protein can be identified by measuring electrophysiological response to application of a specific receptor ligand (Barnard et al. 1982). We injected brain or stomach mRNA into oocytes and examined their electrophysiological responses to the application of different tachykinins. These experiments indicated that substance P and substance K receptors are encoded by different mRNAs, and that these two mRNAs are expressed differentially between the brain and peripheral tissue.

We extended this observation and developed a new cloning strategy to isolate the cDNA clone for bovine substance K receptor (Masu et al. 1987). We size-fractionated bovine stomach mRNA by sucrose gradient centrifugation and constructed a cDNA library from the active fraction of mRNA identified by oocyte expression system. The vector DNA used contained an RNA polymerase promoter next to the insertion site of the cDNA, thus allowing the in vitro mRNA synthesis from the cloned cDNA by a specific RNA polymerase. We identified a cloned cDNA mixture containing a receptor cDNA clone by testing electrophysiologically for the receptor expression following injection of the in vitro synthesized mRNA into the oocyte system. After repeated fractionation of a response-evoking cloned cDNA mixture, we obtained a single cDNA clone that was capable of inducing electrophysiological response to substance K. The sequence analysis of the cloned cDNA indicated that substance K receptor is a polypeptide consisting of 384 amino acid residues. The hydropathicity profile and the sequence data bank analyses revealed that the substance K receptor has seven hydrophobic segments and shares significant sequence similarity with rhodopsin-type receptors comprising adrenergic and muscarinic receptors.

Rhodopsin-type receptors have a structure consisting of seven hydrophobic membrane-spanning domains with an extracellular amino terminus and a cytoplasmic carboxyl terminus (reviewed in Dohlman et al. 1987). As illustrated in figure 2, a similar transmembrane model for substance K receptor can be suggested from its primary structure. The seven putative transmembrane alpha-helices of substance K receptor comprise a continuous stretch of 20-24 uncharged amino acid residues, except that transmembrane segments II, V, and VI contain Asp, His, and His, respectively. The Asp in segment II is conserved for all rhodopsin-type receptors, whereas the presence of the two His residues in the transmembrane domains is characteristic of substance K receptor.

The amino-terminal and carboxyl-terminal regions of substance K receptor show a pattern similar to those of the rhodopsin-type receptors. The amino-terminal region with no signal sequence contains two potential Nglycosylation sites, while the carboxyl-terminal region has many serine and threonine residues as possible phosphorylation sites. These results provide