A proposed bovine neuropeptide Y (NPY) receptor cDNA clone, or its human homologue, confers neither NPY binding sites nor NPY responsiveness on transfected cells

doi:10.1016/0167-0115(93)90392-L

Regulatory Peptides

Volume 47, Issue 3, 22 September 1993, Pages 247-258

https://doi.org/10.1016/0167-0115(93)90392-L Get rights and content

Abstract

Receptors with seven transmembrane domains (7TM) constitute a large family of structurally and functionally related proteins which respond to various types of ligands. We describe here the cloning and expression of a human 7TM receptor, denoted hFB22 (human Fetal Brain 22), which is the homologue (92% amino acid identity) of a bovine receptor (LCR1) reported by others to bind neuropeptide Y (NPY) with a pharmacological profile of the Y3 receptor subtype. However, upon expression in COS1 (confirmed by Northern analysis), COS7 or CHO-K1 cells, the hFB22 receptor did not confer specific ¹²⁵I-Bolton-Hunter-NPY, ³H-propionyl-NPY or ¹²⁵I-peptide YY (PYY) binding sites, in either intact cells or in membrane preparations. Similarly, cells transfected with the corresponding bovine clone (LCR1) did not show specific NPY/PYY binding exceeding that resulting from endogenous binding sites; mock-transfected COS7 cells, used frequently for heterologous expression of receptors, were found to have endogenous specific ¹²⁵I-NPY binding sites ( $B_{max} = 112 fmol/mg$ protein; $K_{d} = 0.25 nM$ ). Moreover, the hFB22 transfected cells, when compared to control transfected cells, did not display de novo NPY- or PYY-induced second messenger responses, i.e., (1) inhibition of forskolin-stimulated cAMP accumulation or (2) ⁴⁵Ca²⁺ influx. The presence of hFB22 mRNA was detected in several human neuroblastoma cell lines, none of which was found to express Y3-like NPY binding sites. hFB22 displays 39% amino acid sequence identity (in the transmembrane regions) to the human interleukin-8 receptor, and 32–36% amino acid identity to the human receptors of angiotensin II, bradykinin, and n-formylpeptide, but only 23% amino acid identity to the previously described human NPY/PYY receptor of the Y1 receptor subtype. Our results show that hFB22 and LCR1 do not encode NPY receptors, and their true ligand(s) remains to be identified.

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The neuropeptide Y₄ receptor (Y₄R), a rhodopsin-like G protein-coupled receptor (GPCR) and the hormone pancreatic polypeptide (PP) are members of the neuropeptide Y family consisting of four receptors (Y₁R, Y₂R, Y₄R, Y₅R) and three highly homologous peptide ligands (neuropeptide Y, peptide YY, PP). In this family, the Y₄R is of particular interest as it is the only subtype with high affinity to PP over NPY. The Y₄R, as a mediator of PP signaling, has a pivotal role in appetite regulation and energy homeostasis, offering potential avenues for the treatment of metabolic disorders such as obesity. PP as anorexigenic peptide is released postprandial from the pancreas in response to food intake, induces satiety signals and contributes to hamper excessive food intake. Moreover, this system was also described to be associated with different types of cancer: overexpression of Y₄R have been found in human adenocarcinoma cells, while elevated levels of PP are related to the development of pancreatic endocrine tumors. The pharmacological relevance of the Y₄R advanced the search for potent and selective ligands for this receptor subtype, which will be significantly progressed through the elucidation of the active state PP-Y₄R cryo-EM structure. This review summarizes the development of novel PP-derived ligands, like Obinepitide as dual Y₂R/Y₄R agonist in clinical trials or UR-AK86c as small hexapeptide agonist with picomolar affinity, as well as the first allosteric modulators that selectively target the Y₄R, e.g. VU0506013 as potent Y₄R positive allosteric modulator or (S)-VU0637120 as allosteric antagonist. Here, we provide valuable insights into the complex physiological functions of the Y₄R and PP and the pharmacological relevance of the system in appetite regulation to open up new avenues for the development of tool compounds for targeted therapies with potential applications in metabolic disorders.
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NPY family receptors include six subtypes (Y1–Y5 and y6) in mammals. The function of Y3 is still controversial,5–7 and the y6 gene is not expressed in primates.8–10 Therefore, most pharmacological studies involving NPY family receptors have been conducted for Y1, Y2, Y4, and Y5 subtypes, which showed that they have unequal distributions and different ligand affinities.11–14
Continuous administration of a 14-amino acid peptide YY (PYY) analog, Ac-[d-Pro²⁴,Pya(4)²⁶,Cha^27,36,Aib^28,31,Lys³⁰]PYY(23–36) (4), which has a high binding affinity and agonist activity for the neuropeptide Y2 receptor (Y2R), has previously shown an antiobesity effect in a 2-week diet-induced obesity (DIO) study in mice. However, there remained a possibility to obtain more potent analogs by further improving its pharmacokinetic profile. A combination of the N-terminal 4-imidazolecarbonyl moiety and three amino acid substitutions, trans-4-hydroxy-d-proline (d-Hyp)²⁴, isovaline (Iva)²⁵, and γ-methylleucine (γMeLeu)²⁸, not only improved the binding affinity of the peptide for Y2R but also increased its anorectic activity in lean mice. In a 2-week DIO study in mice, continuous administration of 4-imidazolecarbonyl-[d-Hyp²⁴,Iva²⁵,Pya(4)²⁶,Cha^27,36,γMeLeu²⁸,Lys³⁰,Aib³¹]PYY(23–36) (31, PYY-1119) at a dose of 0.03 mg/kg/day showed a highly potent antiobesity effect, with more than 10% body weight reduction.
Neuropeptide Y/peptide YY receptor Y2 duplicate in zebrafish with unique introns displays distinct peptide binding properties
2011, Comparative Biochemistry and Physiology - B Biochemistry and Molecular Biology
Citation Excerpt :
Comparisons of a broad range of species have demonstrated that the first jawed vertebrate had two NPY-family peptides, NPY and peptide YY (PYY), and no less than seven receptors. The receptor subtypes are named Y1 through Y8 with Y3 missing (it does not exist as a separate gene product) (Herzog et al., 1993; Jazin et al., 1993). Both the peptide genes and several of the receptor genes were multiplied as a result of two tetraploidizations that took place in early vertebrate evolution (Soderberg et al., 2000; Larhammar et al., 2004; Larsson et al., 2008; Salaneck et al., 2008; Sundström et al., 2008; Larsson et al., 2009), often referred to as 2R for two rounds of genome doubling (Dehal and Boore, 2005; Nakatani et al., 2007; Putnam et al., 2008).
The neuropeptide Y-family peptides and receptors are involved in a broad range of functions including appetite regulation. Both the peptide genes and the receptor genes are known to have duplicated in early vertebrate evolution. The ancestral jawed vertebrate had 7 NPY receptors but the number varies between 4 and 7 in extant vertebrates. Herein we describe the identification of an additional NPY receptor in two fish species, zebrafish and medaka. They cluster together with the Y2 receptors in phylogenetic analyses and seem to be orthologous to each other that is why we have named them Y2-2. Their genes differ from Y2 in having introns in the coding region. Binding studies with zebrafish Y2-2 receptors show that the three endogenous peptides NPY, PYYa and PYYb have similar affinities, 0.15–0.66 nM. This is in contrast to the Y2 receptor where they differed considerably from one another. N-terminally truncated NPY binds poorly and the Y2 antagonist BIIE0246 binds well to Y2-2, results that are reversed in comparison to Y2. Zebrafish Y2-2 mRNA was detected by PCR in the intestine and the eye, but not in the brain. In conclusion, we have found a novel Y2-like NPY/PYY receptor that probably arose in early teleost fish evolution.
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2009, Molecular and Cellular Endocrinology
Laboratory studies consistently demonstrate extended lifespan in animals on calorie restriction (CR), where total caloric intake is reduced by 10–40% but adequate nutrition is otherwise maintained. CR has been further shown to delay the onset and severity of chronic diseases associated with aging such as cancer, and to extend the functional health span of important faculties like cognition. Less understood are the underlying mechanisms through which CR might act to induce such alterations. One theory postulates that CR's beneficial effects are intimately tied to the neuroendocrine response to low energy availability, of which the arcuate nucleus in the hypothalamus plays a pivotal role. Neuropeptide Y (NPY), a neurotransmitter in the front line of the arcuate response to low energy availability, is the primary hunger signal affected by CR and therefore may be a critical mechanism for lifespan extension.
Overview and History of Chemokines and Their Receptors
2005, Current Topics in Membranes
Citation Excerpt :
CXCR3 is also expressed in a subpopulation of neurons in various cortical and subcortical regions of the brain (Coughlan et al., 2000; Xia et al., 2000) and in astrocytes and microglia (Biber et al., 2002). CXCR4 was initially cloned by several laboratories (Federsppiel et al., 1993; Herzog et al., 1993; Jazin et al., 1993; Nomura et al., 1993) and identified as the orphan receptor LESTR by one group of investigators (Loetscher et al., 1994a). This receptor was later identified as fusin, the co‐receptor (with CD4) for syncytium‐inducing laboratory strains of HIV‐1 (Feng et al., 1996).
This chapter provides an overview and the history of chemokines and their receptors. The large family of structurally related chemotactic cytokines involved in the migration and activation of speciﬁc leukocytes is designated as “chemokines.” This family of chemotactic proteins appears to be conserved evolutionarily, as evidenced by its high degree of homology across species from mammals to fish. Chemokines, small molecular weight proteins ranging in size from approximately 8–15 kDa, chemoattract speciﬁc subsets of leukocytes, in addition to other cell types, and are characterized by the position of the ﬁrst two of four cysteine residues in highly conserved positions within their amino (N)‐terminal protein sequence. The chemokine family is now subdivided into four major groups, designated as “CXC” (formerly α chemokines), “CC” (formerly β chemokines), “C” (formerly γ chemokines), and “CX₃C” (formerly δ chemokines). The regulation of leukocyte migration and activation during immune responses is the major function of chemokines; they also modulate additional biological activities, including hematopoiesis, apoptosis, angiogenesis, cell proliferation, and viral pathogenesis.
The Y<inf>1</inf> receptor subtype mediates the cardiovascular changes evoked by NPY administered into the posterior hypothalamic nucleus of conscious rat
2004, Brain Research
Citation Excerpt :
The log(EC50) for each peptide was then plotted against the averaged log's of the reported IC50's and Ki's of each peptide for the Y1, Y2, Y4 and Y5 receptor subtypes [3,10,11,16,19,22,35,37]. Since the Y6 receptor subtype does not exist in the rat [5,32], and the existence of the Y3 receptor is in question and has yet to be cloned [15,17], these two Y receptor subtypes were not included in the correlations. The greatest degree of correlation was found to exist for the Y1 and Y5 receptor subtypes.
An earlier study showed that the neuropeptide Y (NPY) receptor antagonist PYX-2 blocks the enhancement of a carbachol (CCh)-evoked pressor response produced by prior NPY administration into the posterior hypothalamic nucleus (PHN). The Y receptor subtype that mediates this response, and an increase in mean arterial pressure (MAP) and heart rate, remained unknown due to the lack of selectivity of PYX-2 for the Y receptor subtypes. Thus, the present study was undertaken to elucidate the Y receptor subtype responsible for mediating the NPY-evoked cardiovascular responses from the PHN by determining the rank order of potency of several NPY-related peptides for increasing MAP, and by correlating the pressor response evoked by these peptides to reported K_i's and IC₅₀'s for the Y₁, Y₂, Y₄ and Y₅ receptor subtypes. The pharmacological profile (PYY≥NPY≥[Leu³¹,Pro³⁴]NPY≥NPY_13–36≥hPP) and correlations suggest that the Y₁ and/or Y₅ receptor subtypes mediate these cardiovascular changes. Administration of the relatively non-selective Y receptor antagonist PYX-2 or the selective Y₁ receptor antagonist BIBP 3226 into the PHN prior to NPY completely blocked the cardiovascular responses. BIBP 3226 also blocked the cardiovascular changes evoked by [Leu³¹,Pro³⁴]NPY, NPY_13–36 and human pancreatic polypeptide (hPP). In contrast, neither BIBP 3226 nor PYX-2 inhibited the cardiovascular changes induced by peptide YY (PYY) or CCh microinjection into the PHN. These results show that NPY and PYY act on different receptors to mediate their respective cardiovascular changes from the PHN with NPY stimulating the Y₁ receptor.

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Regulatory Peptides

Abstract

References (19)

Receptors for neuropeptide Y: multiple subtypes and multiple second messengers

Trends Pharmacol. Sci.

Characterization of neuropeptide Y binding sites in rat cardiac ventricular membranes

Peptides

Cloning and functional expression of a human neuropeptide Y/peptide YY receptor of the Y1 type

J. Biol. Chem.

Molecular cloning of a G-protein coupled receptor that is highly expressed in lymphocytes and proliferative areas of developing brain

Mol. Cell. Neurosci.

Cloning and characterization of a human angiotensin II type 1 receptor

Biochem. Biophys. Res. Commun.

Cloning, functional expression and developmental regulation of a neuropeptide Y receptor from Drosophila melanogaster

J. Biol. Chem.

Structure/function relationship of proteins belonging to the family of receptors coupled to GTP-binding proteins

Eur. J. Biochem.

Evolution of the neuropeptide Y family of peptides

Neuropeptide Y-related peptides and their receptors: are these receptors potential therapeutic drug targets?

Annu. Rev. Pharmacol. Toxicol.

Cited by (80)

Illuminating the neuropeptide Y<inf>4</inf> receptor and its ligand pancreatic polypeptide from a structural, functional, and therapeutic perspective

Highly potent antiobesity effect of a short-length peptide YY analog in mice

Neuropeptide Y/peptide YY receptor Y2 duplicate in zebrafish with unique introns displays distinct peptide binding properties

Hungry for life: How the arcuate nucleus and neuropeptide Y may play a critical role in mediating the benefits of calorie restriction

Overview and History of Chemokines and Their Receptors

The Y<inf>1</inf> receptor subtype mediates the cardiovascular changes evoked by NPY administered into the posterior hypothalamic nucleus of conscious rat