Diversity in mammalian tachykinin peptidergic neurons: multiple peptides, receptors, and regulatory mechanisms

The tachykinins comprise a family of closely related peptides that participate in the regulation of diverse biological processes. The tachykinin peptides substance P, neurokinin A, neurokinin A(3-10), neuropeptide K, and neuropeptide gamma are produced from a single preprotachykinin gene as a result of differential RNA splicing and differential posttranslational processing. Another tachykinin, neurokinin B, is produced from a separate preprotachykinin gene. These preprotachykinin mRNAs and peptide products are differentially distributed throughout the nervous system. Three distinct G protein-coupled tachykinin receptors exist for these tachykinin peptides. The three receptors interact differentially with the tachykinin peptides and are uniquely distributed throughout the nervous system. The NK-1 receptor preferentially interacts with substance P, the NK-2 receptor prefers neurokinin A, neuropeptide K, and neuropeptide gamma, and the NK-3 receptor interacts best with neurokinin B. Examples of the roles of tachykinin peptidergic neuronal systems are taken from the spinal cord sensory system and the nigrostriatal extrapyramidal motor system. Analysis of the functional significance of multiple tachykinin peptide systems, receptor-second messenger coupling mechanisms, and developmental and regulatory mechanisms underlying peptide mRNA and receptor expression represent areas of current and future investigation.     

New challenges in the study of the mammalian tachykinins

There is an expanding repertoire of mammalian tachykinins produced by a variety of tachykinin genes, gene splicing events and peptide processing. Novel tachykinin-binding molecules/receptors are proposed, but only, three tachykinin receptors are identified with certainty. The question remains - do more tachykinin receptors exist or is there just the need to reappraise our understanding of the known receptors? The tachykinin NK1 receptor, the preferred receptor for both substance P and the peripheral SP-like endokinins, exists in several tissue-specific conformations and isoforms and may provide some clues. This review addresses recent advances in this exciting field and raises challenging new concepts.     

A novel tachykinin-related peptide receptor. Sequence, genomic organization, and functional analysis.

Structurally tachykinin-related peptides have been isolated from various invertebrate species and shown to exhibit their biological activities through a G-protein-coupled receptor (GPCR) for a tachykinin-related peptide. In this paper, we report the identification of a novel tachykinin-related peptide receptor, the urechistachykinin receptor (UTKR) from the echiuroid worm, Urechis unitinctus. The deduced UTKR precursor includes seven transmembrane domains and typical sites for mammalian tachykinin receptors and invertebrate tachykinin-related peptide receptors. A functional analysis of the UTKR expressed in Xenopus oocytes demonstrated that UTKR, like tachykinin receptors and tachykinin-related peptide receptors, activates calcium-dependent signal transduction upon binding to its endogenous ligands, urechistachykinins (Uru-TKs) I-V and VII, which were isolated as Urechis tachykinin-related peptides from the nervous tissue of the Urechis unitinctus in our previous study. UTKR responded to all Uru-TKs equivalently, showing that UTKR possesses no selective affinity with Uru-TKs. In contrast, UTKR was not activated by substance P or an Uru-TK analog containing a C-terminal Met-NH2 instead of Arg-NH2. Furthermore, the genomic analysis revealed that the UTKR gene, like mammalian tachykinin receptor genes, consists of five exons interrupted by four introns, and all the intron-inserted positions are completely compatible with those of mammalian tachykinin receptor genes. These results suggest that mammalian tachykinin receptors and invertebrate tachykinin-related peptide receptors were evolved from a common ancestral GPCR gene. This is the first identification of an invertebrate tachykinin-related peptide receptor from other species than insects and also of the genomic structure of a tachykinin-related peptide receptor gene.     

Retro-all-D and retro isomers of a formyl-methionyl peptide chemoattractant: an insight into the mode of binding at the receptor on rabbit neutrophils

The retro-all-D analog and retro isomer of the formyl-methionyl-carboxamide-tripeptide chemoattractant, CHO-L-Met-L-Leu-L-Phe-NH2, namely CHO-D-Phe-D-Leu-D-Met-NH2 and CHO-L-Phe-L-Leu-L-Met-NH2, respectively, have been synthesized in solution by classical methods and fully characterized. The tetrapeptide CHO-L-Phe-Gly-L-Leu-L-Met-NH2, representing the C-terminal portion of the tachykinin, Substance P, and resembling the sequence of the retro isomer, has also been synthesized and characterized. The three N alpha-formylated tripeptide amides, prepared in order to obtain a deeper insight into the model of binding at the formyl peptide chemotactic receptor on rabbit neutrophils, have been tested for their ability to induce granule enzyme secretion from rabbit peritoneal neutrophils. The retro isomer, CHO-L-Phe-L-Leu-L-Met-NH2 is approximately 100-fold less active, the retro-all-D analog, CHO-D-Phe-D-Leu-D-Met-NH2 approximately 10,000-fold less active and the Substance P analog CHO-L-Phe-Gly-L-Leu-L-Met-NH2 1000-fold less active than the parent formyl peptide chemoattractant, CHO-L-Met-L-Leu-L-Phe-NH2. We interpret these results to indicate that a precise alignment of amino acid side chains as well as backbone amide bonds is an important factor involved in the receptor recognition of the formyl tripeptide chemoattractant.     

Identification and partial characterization of a variant of human CXCR3 generated by posttranscriptional exon skipping

Chemokines are recognized as functionally important in many pathological disorders, which has led to increased interest in mechanisms related to the regulation of chemokine receptor (CKR) expression. Known mechanisms for regulating CKR activity are changes in gene expression or posttranslational modifications. However, little is known about CKR with respect to a third regulatory mechanism, which is observed among other seven-transmembrane receptor subfamilies, the concept of differential splicing or processing of heteronuclear RNA. We now report on the discovery of a variant human CKR, CXCR3, resulting from alternative splicing via exon skipping. The observed RNA processing entails a drastically altered C-terminal protein sequence with a predicted four- or five-transmembrane domain structure, differing from all known functional CKR. However, our data indicate that that this splice variant, which we termed CXCR3-alt, despite its severe structural changes still localizes to the cell surface and mediates functional activity of CXCL11.     

蛋白内含子与蛋白剪接

蛋白内含子和蛋白剪接是蛋白质研究的前沿领域。重点介绍了蛋白内含子的结构和蛋白剪接机理的最新研究成果 ;蛋白内含子如同RNA剪接中的内含子 ,也是一类可移动的遗传元件 ;蛋白内含子目前研究的热点是蛋白内含子的功能研究及其在蛋白质工程和其它生物工程领域的用。     

Direct linkage of three tachykinin receptors to stimulation of both phosphatidylinositol hydrolysis and cyclic AMP cascades in transfected Chinese hamster ovary cells.

The mammalian tachykinin system consists of three distinct peptides, substance P, substance K, and neuromedin K, and possesses three corresponding receptors. In this investigation we examined intracellular signal transduction of the individual tachykinin receptors by transfection and stable expression of these receptor cDNAs in Chinese hamster ovary cells. The three receptors commonly showed a rapid and marked stimulation in both phosphatidylinositol (PI) hydrolysis and cyclic AMP formation in response to tachykinin interaction. Direct linkage of the three receptors to both phospholipase C and adenylate cyclase was evidenced by the finding that tachykinin, added together with GTP, activated these enzyme activities in membrane preparations derived from tachykinin receptor-expressing cells. The stimulation of cyclic AMP formation was less efficient than that of PI hydrolysis in receptor-expressing cells as well as their membrane preparations (about 1 order of magnitude difference in the effective peptide concentrations). However, the stimulatory responses of the PI hydrolysis and cyclic AMP formation in both receptor-expressing cells and their membrane preparations were induced in complete agreement with the tachykinin binding selectivity of each subtype of the receptors. This investigation demonstrated unequivocally that the tachykinin receptors have the potential to couple directly to both phospholipase C and adenylate cyclase and to stimulate PI hydrolysis and cyclic AMP formation.     

Antinociceptive and Met-enkephalin releasing effects of tachykinins and substance P fragments

Substance P (SP), physalaemin, SP4-11, SP5-11 and the SP5-11 analog DiMe-C7 induce an antinociceptive effect in rats after intraventricular administration. Other tachykinins and the N-terminal fragments of SP are inactive. All antinociceptive peptides increase the Met-enkephalin efflux from slices of rat periaqueductal gray matter and their antinociceptive potency is correlated with their capacity to release Met-enkephalin. The results, discussed in the light of current theories on different tachykinin receptors, suggest that the SP-P receptor subtype may be involved in the control of noxious stimulation elicited by SP at supraspinal levels.     

Highly selective agonists for substance P receptor subtypes.

The existence of a third tachykinin receptor (SP-N) in the mammalian nervous system was demonstrated by development of highly selective agonists. Systematic N-methylation of individual peptide bonds in the C-terminal hexapeptide of substance P gave rise to agonists which specifically act on different receptor subtypes. The most selective analog of this series, succinyl-[Asp6,Me-Phe8]SP6-11, elicits half-maximal contraction of the guinea pig ileum through the neuronal SP-N receptor at a concentration of 0.5 nM. At least 60,000-fold higher concentrations of this peptide are required to stimulate the other two tachykinin receptors (SP-P and SP-E). The action of selective SP-N agonists in the guinea pig ileum is antagonized by opioid peptides, suggesting a functional counteraction between opiate and SP-N receptors. These results indicate that the tachykinin receptors are distinct entities which may mediate different physiological functions.     

A novel tachykinin-related peptide receptor of Octopus vulgaris--evolutionary aspects of invertebrate tachykinin and tachykinin-related peptide

The tachykinin (TK) and tachykinin-related peptide (TKRP) family represent one of the largest peptide families in the animal kingdom and exert their actions via a subfamily of structurally related G-protein-coupled receptors. In this study, we have identified a novel TKRP receptor from the Octopus heart, oct-TKRPR. oct-TKRPR includes domains and motifs typical of G-protein-coupled receptors. Xenopus oocytes that expressed oct-TKRPR, like TK and TKRP receptors, elicited an induction of membrane chloride currents coupled to the inositol phosphate/calcium pathway in response to Octopus TKRPs (oct-TKRP I-VII) with moderate ligand selectivity. Substance P and Octopus salivary gland-specific TK, oct-TK-I, completely failed to activate oct-TKRPR, whereas a Substance P analog containing a C-terminal Arg-NH2 exhibited equipotent activation of oct-TKRPs. These functional analyses prove that oct-TKRPs, but not oct-TK-I, serve as endogenous functional ligands through oct-TKRPR, although both of the family peptides were identified in a single species, and the importance of C-terminal Arg-NH2 in the specific recognition of TKRPs by TKRPR is conserved through evolutionary lineages of Octopus. Southern blotting of RT-PCR products revealed that the oct-TKRPR mRNA was widely distributed in the central and peripheral nervous systems plus several peripheral tissues. These results suggest multiple physiologic functions of oct-TKRPs as neuropeptides both in the Octopus central nervous system and in peripheral tissues. This is the first report on functional discrimination between invertebrate TKRPs and salivary gland-specific TKs.     

Peptidylglycine alpha-amidating monooxygenase: a multifunctional protein with catalytic, processing, and routing domains.

Peptide alpha-amidation is a widespread, often essential posttranslational modification shared by many bioactive peptides and accomplished by the products of a single gene encoding a multifunctional protein, peptidylglycine alpha-amidating monooxygenase (PAM). PAM has two catalytic domains that work sequentially to produce the final alpha-amidated product peptide. Tissue-specific alternative splicing can generate forms of PAM retaining or lacking a domain required for the posttranslational separation of the two catalytic activities by endoproteases found in neuroendocrine tissue. Tissue-specific alternative splicing also governs the presence of a transmembrane domain and generation of integral membrane or soluble forms of PAM. The COOH-terminal domain of the integral membrane PAM proteins contains routing information essential for the retrieval of PAM from the surface of endocrine and nonendocrine cells. Tissue-specific endoproteolytic processing can generate soluble PAM proteins from integral membrane precursors. Soluble PAM proteins are rapidly secreted from stably transfected nonneuroendocrine cells but are stored in the regulated secretory granules characteristic of neurons and endocrine cells.     

蛋白质剪接及其在蛋白质工程中的应用

蛋白质剪接是蛋白质内含肽介导的,一种在蛋白质水平上翻译后的加工过程,它由一系列分子内的剪切-连接反应组成。蛋白质内含肽是一个蛋白质前体中的多肽序列,可以催化自身从蛋白质前体中断裂,使两侧的蛋白质外显肽连接成成熟的蛋白质。蛋白质内含肽的发现,不仅丰富了遗传信息翻译后加工的理论,在实践中也有广泛的应用前景。Abstract: Protein splicing , which is an intein mediated posttranslational processing, involves a series of intramolecular cleavage-ligation reactions. Intein is an intervening polypeptide which can catalytic self-cleavage from a pre-protein accompanied by the concomitant joining of the two flanking polypeptides (the extein) through a peptide bond. Protein splicing not only enriches genetic theory of posttranslational processing, but also have wide application prospect.     

Characterization of endothelin-converting enzyme-2. Implication for a role in the nonclassical processing of regulatory peptides

Most neuroendocrine peptides are generated by proteolysis of the precursors at basic residue cleavage sites. Prohormone convertases belonging to the subtilisin family of serine proteases are primarily responsible for processing at these "classical sites." In addition to the classical cleavages, a subset of bioactive peptides is generated by processing at "nonclassical" sites. The proteases responsible for these cleavages have not been well explored. Members of several metalloprotease families have been proposed to be involved in nonclassical processing. Among them, endothelin-converting enzyme-2 (ECE-2) is a good candidate because it exhibits a neuroendocrine distribution and an acidic pH optimum. To examine the involvement of this protease in neuropeptide processing, we purified the recombinant enzyme and characterized its catalytic activity. Purified ECE-2 efficiently processes big endothelin-1 to endothelin-1 by cleavage between Trp(21) and Val(22) at acidic pH. To characterize the substrate specificity of ECE-2, we used mass spectrometry with a panel of 42 peptides as substrates to identify the products. Only 10 of these 42 peptides were processed by ECE-2. A comparison of residues around the cleavage site revealed that ECE-2 exhibits a unique cleavage site selectivity that is related to but distinct from that of ECE-1. ECE-2 tolerates a wide range of amino acids in the P1-position and prefers aliphatic/aromatic residues in the P1'-position. However, only a small fraction of the aliphatic/aromatic amino acid-containing sites were cleaved, indicating that there are additional constraints beyond the P1- and P1'-positions. The enzyme is able to generate a number of biologically active peptides from peptide intermediates, suggesting an important role for this enzyme in the biosynthesis of regulatory peptides. Also, ECE-2 processes proenkephalin-derived bovine adrenal medulla peptides, and this processing leads to peptide products known to have differential receptor selectivity. Finally, ECE-2 processes PEN-LEN, an endogenous inhibitor of prohormone convertase 1, into products that do not inhibit the enzyme. Taken together, these results are consistent with an important role for ECE-2 in the processing of regulatory peptides at nonclassical sites.     

Functional characterization on invertebrate and vertebrate tissues of tachykinin peptides from octopus venoms

It has been previously shown that octopus venoms contain novel tachykinin peptides that despite being isolated from an invertebrate, contain the motifs characteristic of vertebrate tachykinin peptides rather than being more like conventional invertebrate tachykinin peptides. Therefore, in this study we examined the effect of three variants of octopus venom tachykinin peptides on invertebrate and vertebrate tissues. While there were differential potencies between the three peptides, their relative effects were uniquely consistent between invertebrate and vertebrae tissue assays. The most potent form (OCT-TK-III) was not only the most anionically charged but also was the most structurally stable. These results not only reveal that the interaction of tachykinin peptides is more complex than previous structure–function theories envisioned, but also reinforce the fundamental premise that animal venoms are rich resources of novel bioactive molecules, which are useful investigational ligands and some of which may be useful as lead compounds for drug design and development.