Modeling of Neuropeptide Receptors Y1, Y4, Y5, and Docking Studies with Neuropeptide Antagonist Analogues: Implications for Selectivity

Abstract

Neuropeptide Y (NPY), receptors belong to the G-protein coupled receptor superfamily. NPY mediates several physiological responses, such as blood pressure, food intake, sedation. These actions of NPY are mediated by six receptor subtypes denoted as Y₁-Y₅ and y₆. Modeling of receptor subtypes and binding site identification is an important step in developing new therapeutic agents. We have attempted to model the three NPY receptor types, Y1, Y4, and Y5 using homology modeling and threading methods. The models are consistent with previously reported experimental evidence. To understand the interaction and selectivity of NPY analogues with different neuropeptide receptors, docking studies of two neuropeptide analogues (BVD10 and BVD15) with receptors Y1 and Y4 were carried out. Results of the docking studies indicated that the interaction of ligands BVD10 and BVD15 with Y1 and Y4 receptors are different. These results were evaluated for selectivity of peptide analogues BVD10 and BVD15 towards the receptors.     

Different mode of arrestin-3 binding at the human Y1 and Y2 receptor

GPCR internalization, which is induced by arrestin recruitment, is an important mechanism for the regulation of signaling and receptor quantity at the cell surface.In this study, differences in the mechanism of arrestin-3 (arr-3) recruitment to the neuropeptide Y₁ and Y₂ receptor were identified. These receptors play an essential role in the regulation of feeding, energy homeostasis and cancer. The Y₁R displays high affinity to arr-3, which induces rapid internalization of the arrestin/receptor complex. In contrast, the Y₂R has a lower affinity for arr-3. Internalization is induced by arrestin binding, but arr-3 is released from the receptor and remains at the membrane while the receptor internalizes. Moreover, the deletion of the finger loop region of arr-3 reduces its agonist-dependent recruitment to the Y₂R significantly, but not to the Y₁R suggesting different binding conformations.For the first time, the formation of a supercomplex consisting of Y receptor, Gα₀ protein and arrestin was studied by BRET-assay. We demonstrated that the Y₁R is able to bind Gα₀ protein as well as arr-3 simultaneously and internalizes as a supercomplex. For the Y₂R no supercomplex formation was observed.By substituting the C-terminus or specific residues within the intracellular loop 1 and 2 of the receptors, the arr-3 recruitment of the Y₁R and Y₂R can be switched. Thus, we shed light on the specific spatio-temporal distribution of Gα₀ protein and arrestin in response to Y₁ versus Y₂ receptor activation and identified the molecular determinants.     

Conformational Analysis of Neuropeptide Y Segments by CD,NMR Spectroscopy and Restrained Molecular Dynamics

Neuropeptide Y (NPY), a peptide amide comprising 36 residue has been shown to act as a potent vasoconstrictor. In order to shed light on the structural requirements for the biological activities with respect to the different prerequisites for affinity to the NPY receptor subtypes Y₁ and Y₂, in the present study the syntheses and conformational analyses of two C-terminal segments, NPY(18–36) and NPY(13–36), are described. The results obtained by CD measurements, two-dimensional NMR spectros copy and a conformational refinement of the NMR-derived structure by molecular mechanics simulations support the findings of previously published structure –activity relationship studies for biologically active and selective compounds. In particular, the α-helical conformation as well as an appropriate exposure of the side chains of the critical C-terminal dipeptide within NPY(18–36) are in agreement with the prerequisites proposed for Y₂ receptor binding of that segment.     

Development of selective agonists and antagonists of P2Y receptors

Although elucidation of the medicinal chemistry of agonists and antagonists of the P2Y receptors has lagged behind that of many other members of group A G protein-coupled receptors, detailed qualitative and quantitative structure–activity relationships (SARs) were recently constructed for several of the subtypes. Agonists selective for P2Y₁, P2Y₂, and P2Y₆ receptors and nucleotide antagonists selective for P2Y₁ and P2Y₁₂ receptors are now known. Selective nonnucleotide antagonists were reported for P2Y₁, P2Y₂, P2Y₆, P2Y₁₁, P2Y₁₂, and P2Y₁₃ receptors. At the P2Y₁ and P2Y₁₂ receptors, nucleotide agonists (5′-diphosphate derivatives) were converted into antagonists of nanomolar affinity by altering the phosphate moieties, with a focus particularly on the ribose conformation and substitution pattern. Nucleotide analogues with conformationally constrained ribose-like rings were introduced as selective receptor probes for P2Y₁ and P2Y₆ receptors. Screening chemically diverse compound libraries has begun to yield new lead compounds for the development of P2Y receptor antagonists, such as competitive P2Y₁₂ receptor antagonists with antithrombotic activity. Selective agonists for the P2Y₄, P2Y₁₁, and P2Y₁₃ receptors and selective antagonists for P2Y₄ and P2Y₁₄ receptors have not yet been identified. The P2Y₁₄ receptor appears to be the most restrictive of the class with respect to modification of the nucleobase, ribose, and phosphate moieties. The continuing process of ligand design for the P2Y receptors will aid in the identification of new clinical targets.     

Molecular characterization of the ligand–receptor interaction of the neuropeptide Y family

Neuropeptide Y (NPY), peptide YY (PYY) and pancreatic polypeptide (PP) belong to the NPY hormone family and activate a class of receptors called the Y‐receptors, and also belong to the large superfamily of the G‐protein coupled receptors. Structure–affinity and structure–activity relationship studies of peptide analogs, combined with studies based on site‐directed mutagenesis and anti‐receptor antibodies, have given insight into the individual characterization of each receptor subtype relative to its interaction with the ligand, as well as to its biological function. A number of selective antagonists at the Y₁‐receptor are available whose structures resemble that of the C‐terminus of NPY. Some of these compounds, like BIBP3226, BIBO3304 and GW1229, have recently been used for in vivo investigations of the NPY‐induced increase in food intake. Y₂‐receptor selective agonists are the analog cyclo‐(28/32)‐Ac‐[Lys²⁸‐Glu³²]‐(25–36)‐pNPY and the TASP molecule containing two units of the NPY segment 21–36. Now the first antagonist with nanomolar affinity for the Y₂‐receptor is also known, BIIE0246. So far, the native peptide PP has been shown to be the most potent ligand at the Y₄‐receptor. However, by the design of PP/NPY chimera, some analogs have been found that bind not only to the Y₄‐, but also to the Y₅‐receptor with subnanomolar affinities, and are as potent as NPY at the Y₁‐receptor. For the characterization of the Y₅‐receptor in vitro and in vivo, a new class of highly selective agonists is now available. This consists of analogs of NPY and of PP/NPY chimera which all contain the motif Ala³¹‐Aib³². This motif has been shown to induce a 3₁₀‐helical turn in the region 28–31 of NPY and is suggested to be the key motif for high Y₅‐receptor selectivity. The results of feeding experiments in rats treated with the first highly specific Y₅‐receptor agonists support the hypothesis that this receptor plays a role in the NPY‐induced stimulation of food intake. In conclusion, the selective compounds for the different Y‐receptor subtypes known so far are promising tools for a better understanding of the physiological properties of the hormones of the NPY family and related receptors. Copyright © 2000 European Peptide Society and John Wiley & Sons, Ltd.     

Dimeric argininamide-type neuropeptide Y receptor antagonists: Chiral discrimination between Y1 and Y4 receptors

The structurally related peptides neuropeptide Y (NPY), peptide YY (PYY) and pancreatic polypeptide (PP) are endogenous agonists of the NPY receptor (YR) family, which in humans comprises four functionally expressed subtypes, designated Y₁R, Y₂R, Y₄R and Y₅R. Nonpeptide antagonists with high affinity and selectivity have been described for the Y₁R, Y₂R and Y₅R, but such compounds are still lacking for the Y₄R. In this work, the structures of the high affinity selective (R)-argininamide-type Y₁R antagonists BIBP3226 and BIBO3304 were linked via the guanidine or urea moieties to give homo-dimeric argininamides with linker lengths ranging from 31 to 41 atoms. Interestingly, the twin compounds proved to be by far less selective for the Y₁R than the R-configured monovalent parent compounds. The decrease in selectivity ratio was most pronounced for Y₁R versus Y₄R subtype, resulting in comparable affinities of bivalent ligands for Y₁R and Y₄R (e.g. UR-MK177 ((R,R)-49): K_i = 230 nM (Y₁R) and 290 nM (Y₄R)). With a K_i value of 130 nM and a K_b value of 20 nM, UR-MK188 ((R,R)-51) was superior to all Y₄R antagonists known to date. The S,S-configured optical antipodes of UR-MK177 and UR-MK188 (UR-MEK381 ((S,S)-49) and UR-MEK388 ((S,S)-51)) were synthesized to investigate the stereochemical discrimination by the different receptor subtypes. Whereas preference for R,R-configured argininamides was characteristic of the Y₁R, stereochemical discrimination by the Y₄R was not observed. This may pave the way to selective Y₄R antagonists.     

Prokaryotic expression,in vitro folding,and molecular pharmacological characterization of the neuropeptide Y receptor type 2

G protein‐coupled receptors (GPCRs) are a class of membrane proteins that represent a major target for pharmacological developments. However, there is still little knowledge about GPCR structure and dynamics since high‐level expression and characterization of active GPCRs in vitro is extremely complicated. Here, we describe the recombinant expression and functional folding of the human Y₂ receptor from inclusion bodies of E. coli cultures. Milligram protein quantities were produced using high density fermentation and isolated in a single step purification with a yield of over 20 mg/L culture. Extensive studies were carried out on in vitro refolding and stabilization of the isolated receptor in detergent solution. The specific binding of the ligand, the 36 residue neuropeptide Y (NPY), to the recombinant Y₂ receptors in micellar form was shown by several radioligand affinity assays. In competition experiments, an IC₅₀ value in low nanomolar range could be determined. Further, a K_D value of 1.9 nM was determined from a saturation assay, where NPY was titrated to the recombinant Y₂ receptors. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Regulation of P2Y1 Receptor Traffic by Sorting Nexin 1 is Retromer Independent

The activity and traffic of G‐protein coupled receptors (GPCRs) is tightly controlled. Recent work from our laboratory has shown that P2Y₁ and P2Y₁₂ responsiveness is rapidly and reversibly modulated in human platelets and that the underlying mechanism requires receptor trafficking as an essential part of this process. However, little is known about the molecular mechanisms underlying P2Y receptor traffic. Sorting nexin 1 (SNX1) has been shown to regulate the endosomal sorting of cell surface receptors either to lysosomes where they are downregulated or back to the cell surface. These functions may in part be due to interactions of SNX1 with the mammalian retromer complex. In this study, we investigated the role of SNX1 in P2Y receptor trafficking. We show that P2Y₁ receptors recycle via a slow recycling pathway that is regulated by SNX1, whereas P2Y₁₂ receptors return to the cell surface via a rapid route that is SNX1 independent. SNX1 inhibition caused a dramatic increase in the rate of P2Y₁ receptor recycling, whereas inhibition of Vps26 and Vps35 known to be present in retromer had no effect, indicating that SNX1 regulation of P2Y₁ receptor recycling is retromer independent. In addition, inhibition of SNX4, 6 and 17 proteins did not affect P2Y₁ receptor recycling. SNX1 has also been implicated in GPCR degradation; however, we provide evidence that P2Y receptor degradation is SNX1 independent. These data describe a novel function of SNX1 in the regulation of P2Y₁ receptor recycling and suggest that SNX1 plays multiple roles in endocytic trafficking of GPCRs.     

Structural and functional evolution of the P2Y12-like receptor group

Metabotropic pyrimidine and purine nucleotide receptors (P2Y receptors) belong to the superfamily of G protein-coupled receptors (GPCR). They are distinguishable from adenosine receptors (P1) as they bind adenine and/or uracil nucleotide triphosphates or diphosphates depending on the subtype. Over the past decade, P2Y receptors have been cloned from a variety of tissues and species, and as many as eight functional subtypes have been characterized. Most recently, several members of the P2Y₁₂-like receptor group, which includes the clopidogrel-sensitive ADP receptor P2Y₁₂, have been deorphanized. The P2Y₁₂-like receptor group comprises several structurally related GPCR which, however, display heterogeneous agonist specificity including nucleotides, their derivatives, and lipids. Besides the established function of P2Y₁₂ in platelet activation, expression in macrophages, neuronal and glial cells as well as recent results from functional studies implicate that several members of this group may have specific functions in neurotransmission, inflammation, chemotaxis, and response to tissue injury. This review focuses specifically on the structure-function relation and shortly summarizes some aspects of the physiological relevance of P2Y₁₂-like receptor members.     

Determination of Affinity and Activity of Ligands at the Human Neuropeptide Y Y4 Receptor by Flow Cytometry and Aequorin Luminescence

Fluorescence-labeled neuropeptide Y (NPY) has been used in flow cytometric binding assays for the determination of affinity constants of NPY Y₁, Y₂, and Y₅ receptor ligands. Because the binding of fluorescent NPY is insufficient for competition studies at the human Y₄ receptor (hY₄R), we replaced Glu-4 in hPP with Lys for the derivatization with cyanine-5. Because cy5-[K⁴]hPP has high affinity (K_d 5.6 nM) to the hY₄R, it was used as a probe in a flow cytometric binding assay. Specific binding of cy5-[K⁴]hPP to hY₄R was visualized by confocal microscopy. The hY₄R, the chimeric G protein G_qi5 and mitochondrially targeted apoaequorin were stably coexpressed in CHO cells. Aequorin luminescence was quantified in a microplate reader and by a CCD camera. By application of these methods 3-cyclohexyl-N-[(3-1H-imidazol-4-ylpropylamino)(imino)methyl]propanamide (UR-AK49) was discovered as the first nonpeptidic Y₄R antagonist (pK_i 4.17), a lead to be optimized in terms of potency and selectivity.     

Differential expression of P2Y receptors in the rat cochlea during development

Purinergic signaling has broad physiological significance to the hearing organ, involving signal transduction via ionotropic P2X receptors and metabotropic G-protein-coupled P2Y and P1 (adenosine), alongside conversion of nucleotides and nucleosides by ecto-nucleotidases and ecto-nucleoside diphosphokinase. In addition, ATP release is modulated by acoustic overstimulation or stress and involves feedback regulation. Many of these principal elements of the purinergic signaling complex have been well characterized in the cochlea, while the characterization of P2Y receptor expression is emerging. The present study used immunohistochemistry to evaluate the expression of five P2Y receptors, P2Y₁, P2Y₂, P2Y₄, P2Y₆, and P2Y₁₂, during development of the rat cochlea. Commencing in the late embryonic period, the P2Y receptors studied were found in the cells lining the cochlear partition, associated with establishment of the electrochemical environment which provides the driving force for sound transduction. In addition, early postnatal P2Y₂ and P2Y₄ protein expression in the greater epithelial ridge, part of the developing hearing organ, supports the view that initiation and regulation of spontaneous activity in the hair cells prior to hearing onset is mediated by purinergic signaling. Sub-cellular compartmentalization of P2Y receptor expression in sensory hair cells, and diversity of receptor expression in the spiral ganglion neurons and their satellite cells, indicates roles for P2Y receptor-mediated Ca²⁺-signaling in sound transduction and auditory neuron excitability. Overall, the dynamics of P2Y receptor expression during development of the cochlea complement the other elements of the purinergic signaling complex and reinforce the significance of extracellular nucleotide and nucleoside signaling to hearing.     

Neuropeptide Y, an orexigenic hormone, regulates phagocytic activity of lizard splenic phagocytes

Present in vitro study in the wall lizard Hemidactylus flaviviridis, for the first time in ectothermic vertebrates, demonstrated the immunoregulatory role of neuropeptide Y (NPY) and its receptor-coupled downstream signaling cascade. NPY inhibited the percentage phagocytosis and phagocytic index of splenic phagocytes. The inhibitory effect of NPY on phagocytosis was completely antagonized by Y₂ and Y₅ receptor antagonists. This suggests that NPY mediated its effect on phagocytosis through Y₂ and Y₅ receptors. Further, NPY receptor-coupled downstream signaling cascade for NPY effect on phagocytosis was explored using the inhibitors of adenylate cyclase (SQ 22536) and protein kinase A (H-89). The SQ 22536/H-89 in a concentration-related manner decreased the inhibitory effect of NPY on phagocytosis. Further, an increase in intracellular cAMP level was observed in response to NPY. Taken together, it can be concluded that NPY via Y₂ and Y₅ receptor-coupled AC-cAMP-PKA pathway downregulated the phagocytic activity of lizard splenic phagocytes.     

The cloned guinea pig neuropeptide Y receptor Y1 conforms to other mammalian Y1 receptors.

We have cloned the guinea pig neuropeptide Y (NPY) Y1 receptor and found it to be 92-93% identical to other cloned mammalian Y1 receptors. Porcine NPY and peptide YY (PYY) displayed affinities of 43 pM and 48 pM, respectively. NPY2-36 and NPY3-36 had 6- and 46-fold lower affinity, respectively, than intact NPY. Functional coupling was measured by using a microphysiometer. Human NPY and PYY were equipotent in causing extracellular acidification with EC50 values of 0.59 nM and 0.69 nM, respectively, whereas NPY2-36 and NPY3-36 were about 15-fold and 500-fold less potent, respectively, than NPY. The present study shows that the cloned guinea pig Y1 receptor is very similar to its orthologues in other mammals, both with respect to sequence and pharmacology. Thus, results from previous studies on guinea pig NPY receptors might imply the existence of an additional Y1-like receptor sensitive to B1BP3226.     

Modeling P2Y receptor-Ca response coupling in taste cells

Here we elaborated an analytical approach for the simulation of dose-response curves mediated by cellular receptors coupled to PLC and Ca²⁺ mobilization. Based on a mathematical model of purinergic Ca²⁺ signaling in taste cells, the analysis of taste cells responsiveness to nucleotides was carried out. Consistently with the expression of P2Y₂ and P2Y₄ receptors in taste cells, saturating ATP and UTP equipotently mobilized intracellular Ca²⁺. Cellular responses versus concentration of BzATP, a P2Y₂ agonist and a P2Y₄ antagonist, implicated high and low affinity BzATP receptors. Suramin modified the BzATP dose-response curve in a manner that suggested the low affinity receptor to be weakly sensitive to this P2Y antagonist. Given that solely P2Y₂ and P2Y₁₁ are BzATP receptors, their high sensitivity to suramin is poorly consistent with the suramin effects on BzATP responses. We simulated a variety of dose-response curves for different P2Y receptor sets and found that the appropriate fit of the overall pharmacological data was achievable only with dimeric receptors modeled as P2Y₂/P2Y₄ homo- and heterodimers. Our computations and analytical analysis of experimental dose-response curves raise the possibility that ATP responsiveness of mouse taste cells is mediated by P2Y₂ and P2Y₄ receptors operative mostly in the dimeric form.     

Fluorescent Labelled Analogues of Neuropeptide Y for the Characterization of Cells Expressing NPY Receptor Subtypes

Abstract

Porcine neuropeptide Y (NPY), a 36 amino acid hormone of the pancreatic polypeptide family, and subtype selective analogues have been synthesized by solid phase peptide synthesis. The peptides were labelled with Cy3^TM, a commercially available fluorescent marker based on a cyanine dye, by solid phase strategy. During the cleavage α partial fragmentation of the fluorescent marker occurred. This has been investigated by means of HPLC and electrospray mass spectrometry. The labelled analogues of NPY showed high affinity to the NPY receptor subtypes Y₁ and Y₂. Thus, Cy3-NPY. Y₁-selective Cy3-[Pro³⁴] NPY and Y₂ selective Cy3-[Ahx^5–24] NPY were used to label SK-N-MC- and SMS-KAN-cells, which are stably expressing the Y₁-(SK-N-MC) and the Y₂-receptor subtype (SMS-KAN). The binding of the labelled analogues to the receptors was reversible and specific. The photoactivatable analogue, [(Tmd)Phe²⁷] NPY, which showed high affinity to both receptor subtypes was labelled with Cy3 in solution. Whereas the fluorescent labelling of the cells with analogues without photoactivatable amino acid was reversible, successful photocrosslinking could be investigated by the irreversible staining of the cells using Cy3-[(Tmd)Phe²⁷] NPY. These subtype selective analogues are exciting tools to trace receptors in tissues and to identify the pharmacologically characterized subtypes without radioactivity.     

Molecular Modeling of the NPY Binding Site on the Y1 Receptor

A three-dimensional model of the neuropeptide Y (NPY) - rat Y₁ (rY₁) receptor complex and of the NPY _13-36 - rY₁ receptor complex was constructed by molecular modeling based on the electron density projection map of rhodopsin and on site-directed mutagenesis studies of neuropeptide receptors. In order to further guide the modeling, the nucleotide sequences encoding Trp287, Cys295 and His297 in the third extracellular loop of the rY₁ receptor, were altered by site-directed mutagenesis experiments. Single-point mutated receptors were expressed in COS-7 cells, and tested for their ability to bind radio labelled NPY (³H-NPY). Mutations of Trp287 and His297 completely abolished binding of ³H-NPY. The Cys295Ser mutation only slightly decreased the binding of ³H-NPY, suggesting that the involvement of Cys295 in a disulphide bond is not essential for maintaining the correct three-dimensional structure of the binding site for NPY. Molecular dynamics simulations of NPY-rY₁ receptor interactions suggested that Asp199, Asp103 and Asp286 in the receptor interact, respectively, with Lys4, Arg33 and Arg35 of NPY. The simulations also suggested that His297 acts as a hydrogen acceptor from Arg35 in NPY, and that Tyr1 of NPY interacts with a binding pocket on the receptor formed by Asn115, Asp286, Trp287 and His297. Tyr36 in NPY interacted both with Thr41 and Tyr99 via hydrogen bonds, and also with Asn296, His297 and Phe301. The present study suggests that amino acid residues at the extracellular end of the transmembrane helices and in the extracellular loops are strongly involved in binding to NPY and NPY_13-36.Electronic Supplementary Material available.     

Distribution of P2Y<Subscript>2</Subscript> receptors in the guinea pig enteric nervous system and its coexistence with P2X<Subscript>2</Subscript> and P2X<Subscript>3</Subscript> receptors,neuropeptide Y,nitric oxide synthase and calretinin

The distribution of P2Y₂ receptor-immunoreactive (ir) neurons and fibers and coexistence of P2Y₂ with P2X₂ and P2X₃ receptors, neuropeptide Y (NPY), calretinin (CR), calbindin (CB) and nitric oxide synthase (NOS) was investigated with immunostaining methods. The results showed that P2Y₂-ir neurons and fibers were distributed widely in myenteric and submucous plexuses of the guinea pig stomach corpus, jejunum, ileum and colon. The typical morphology of P2Y₂-ir neurons was a long process with strong positive staining on the same side of the cell body. The P2Y₂-ir neurons could be Dogiel type 1. About 40–60% P2X₃-ir neurons were immunoreactive for P2Y₂ in the myenteric plexus and all the P2X₃-ir neurons expressed the P2Y₂ receptor in the submucosal plexus; almost all the NPY-ir neurons and the majority of CR-ir neurons were also immunoreactive for P2Y₂, especially in the myenteric plexus of the small intestine; no P2Y₂-ir neurons were immunoreactive for P2X₂ receptors, CB and NOS. It is shown for the first time that S type/Dogiel type 1 neurons with fast P2X and slow P2Y receptor-mediated depolarizations could be those neurons expressing both P2Y₂-ir and P2X₃-ir and that they are widely distributed in myenteric and submucosal plexuses of guinea pig gut.     

Immunohistochemical distribution of neuropeptide Y,peptide YY,pancreatic polypeptide-like immunoreactivity and their receptors in the epidermal skin of healthy women

Few studies have suggested that neuropeptide Y (NPY) could play an important role in skin functions. However, the expression of NPY, the related peptides, peptide YY (PYY) and pancreatic polypeptide (PP) and their receptors have not been investigated in human skin. Using specific antisera directed against NPY, PYY, PP and the Y₁, Y₂, Y₄ and Y₅ receptor subtypes, we investigated here the expression of these markers. NPY-like immunoreactivity (ir) in the epidermal skin could not be detected. For the first time we report the presence of positive PP-like ir immunofluorescent signals in epidermal cells, i.e. keratinocytes of skin from three areas (abdomen, breast and face) obtained as surgical left-overs. The immunofluorescent signal of PP-like ir varies from very low to high level in all three areas. In contrast, PYY-like ir is only expressed in some cells and with varied level of intensity. Furthermore and for the first time we observed specific Y₁ and Y₄ receptor-like ir in all epidermal layers, while the Y₂ and Y₅ subtypes were absent. Interestingly, as seen in human epidermis, in Episkin, a reconstituted human epidermal layer, we detected the presence of PP-like as well as Y₁-like and Y₄-like ir. These data have shown the presence and distribution of PYY, PP and Y₁ and Y₄ receptors in the human skin and Episkin, suggesting possible novel roles of NPY related peptides and their receptors in skin homeostasis.     

The P2Y4 receptor forms homo-oligomeric complexes in several CNS and PNS neuronal cells

It is well established that several cell surface receptors interact with each other to form dimers and oligomers, which are essential for their activation. Since little is known about the quaternary structure of P2Y receptors, in the present work, we investigated the expression of the G-protein-coupled P2Y₄ subunit as monomeric or higher-order complex protein. We examined both endogenously expressed P2Y₄ subtype with the aid of specific anti-P2Y₄ antiserum, and heterologously transfected P2Y₄-tagged receptors with the use of antitag antibodies. In both cases, we found the P2Y₄ receptor displaying molecular masses corresponding to monomeric, dimeric and oligomeric structures. Experiments performed in the absence of reducing agents demonstrated that there is a strict correlation among the multiple protein bands and that the multimeric forms are at least partially assembled by disulphide bonds. The direct demonstration of P2Y₄ homodimerisation comes instead from co–transfection and differential co–immunoprecipitation experiments, with the use of differently tagged P2Y₄ receptors and antitag antibodies. The structural propensity of the P2Y₄ protein to form homo-oligomers may open the possibility of a novel regulatory mechanism of physiopathological functions for this and additional P2Y receptors.