Cloning and characterization of Rhesus monkey neuropeptide Y receptor subtypes

Gehlert, Donald R.; Yang, Pinchen; George, Carolyn; Wang, Yi; Schober, Douglas A.; Gackenheimer, Susan L.; Johnson, Dwayne; Beavers, Lisa S.; Gadski, Robert A.; Baez, Melvyn

doi:10.1016/s0196-9781(01)00336-9

Cited by 19 publications

(18 citation statements)

References 29 publications

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“…It was found that the affinities of the radioligands did not differ more than 3-fold from the wild-type receptors ( Fig. 1; Gehlert et al, 2001). This is in agreement with findings for the human -opioid receptor where tagging the receptor with Rluc did not affect radioligand binding affinity (Ramsay et al, 2002).…”

Section: Discussionsupporting

confidence: 87%

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The Use of Bioluminescence Resonance Energy Transfer 2 to Study Neuropeptide Y Receptor Agonist-Induced β-Arrestin 2 Interaction

Berglund¹,

Schober²,

Statnick³

et al. 2003

J Pharmacol Exp Ther

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The neuropeptide Y (NPY) family peptides NPY, peptide YY (PYY), and pancreatic polypeptide (PP) bind to four G proteincoupled receptors (GPCRs): Y1, Y2, Y4, and Y5. A key step in the desensitization and internalization of GPCRs is the association of the receptor with ␤-arrestins. In the present study, these receptors were analyzed with respect to their ability to interact with GFP2-tagged ␤-arrestin 2 using the new bioluminescence resonance energy transfer 2 method. Agonists induced a concentration-dependent association of ␤-arrestin 2 with all four receptors. Whereas the Y1 receptor exhibited the highest maximum response and rapid association (t 1/2 ϭ 3.4 min), the maximal signals for the association of Y2 and Y4 receptors were less than half of that of Y1, and the association rates were much slower. Interestingly, when evaluated at the Y4 receptor, the Y4 agonist 1229U91 [(Ile,Glu,Pro,Dpr,Tyr,Arg, Leu,Arg,Try-NH 2 )-2-cyclic(2,4Ј),(2Ј,4)-diamide] was unable to provoke the same maximal response as human PP, suggesting that 1229U91 is a partial agonist. When stimulated by PYY, the Y5 receptor responded with a t 1/2 of 4.6 min and a maximal response approximately 60% of what was observed with Y1. Because ␤-arrestins are key components in GPCR internalization, it is interesting to note that the receptor that is known to internalize rapidly (Y1) exhibits the most rapid association with ␤-arrestin 2, whereas the receptor that is known to internalize slowly, or not at all (Y2) associates slowly with ␤-arrestin 2.

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Section: Discussionsupporting

confidence: 87%

“…The cloning of the rhesus Y1, Y2, and Y5 receptors has been described elsewhere (Gehlert et al, 2001). To clone the rhesus Y4 receptor, two primers based on the 5Ј-and 3Ј-untranslated sequences of the human Y4 receptor gene were synthesized.…”

Section: Methodsmentioning

confidence: 99%

The Use of Bioluminescence Resonance Energy Transfer 2 to Study Neuropeptide Y Receptor Agonist-Induced β-Arrestin 2 Interaction

Berglund¹,

Schober²,

Statnick³

et al. 2003

J Pharmacol Exp Ther

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“…Our group has recently cloned the Y1, Y2, Y5 (Gehlert et al, 2001), and Y4 (Berglund et al, 2003b) receptors from the rhesus monkey. In this paper, we discuss the detailed characterization of the rhY4 receptor with regards to pharmacology and dimerization.…”

Section: Discussionmentioning

confidence: 99%

“…The Y1, Y2, and Y5 receptors from the rhesus monkey have been cloned previously (Gehlert et al, 2001). Using clones containing the coding sequence of the rhesus Y1 and Y2 receptors and the same genomic DNA as above as template for the rhY5 receptor and the primer pairs: rhY1.fH (5Ј-AAGCTTAAGCTTACCATGAATTCAA-CATTATTTTCCCAG-3Ј) and rhY1.NSrKS (5Ј-CCGCGGTACCG-ATTCTTTCATTATCATCATTGTTG-3Ј); rhY2.fH (5Ј-AAGCTTAA-GCTTACCATGGGTCCAATAGGTACAGAGG-3Ј) and rhY2NSrKS (5Ј-CCGCGGTACCGACATTGGTAGCTTCTGTGAAAG-3Ј); rhY5.fH (5Ј-AAGCTTAAGCTTACCATGGATTTAGAGCTCGATGAAT-3Ј) and rhY5.NSrKS (5Ј-CCGCGGTACCCATATGAAGACAGTG-TATAAGGGAC), the coding sequence of these receptors were amplified by PCR and cut and ligated into plasmid pRLUC-N2 as described for rhY4, generating the constructs rhY1-RLUC, rhY2-RLUC, and rhY5-RLUC.…”

Section: Methodsmentioning

confidence: 99%

Neuropeptide Y Y4 Receptor Homodimers Dissociate upon Agonist Stimulation

Berglund¹,

Schober²,

Esterman³

et al. 2003

J Pharmacol Exp Ther

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The pancreatic polypeptide-fold family of peptides consists of three 36-amino acid peptides, namely neuropeptide Y (NPY), peptide YY, and pancreatic polypeptide (PP). These peptides regulate important functions, including food intake, circadian rhythms, mood, blood pressure, intestinal secretion, and gut motility, through four receptors: Y1, Y2, Y4, and Y5. Additional receptor subtypes have been proposed based on pharmacology observed in native tissues. Recent studies with other Gprotein-coupled receptors have shown that homo-and heterodimerization may be important in determining receptor function and pharmacology. In the present study, the recently cloned rhesus (rh) Y4 receptor was evaluated using radioligand binding, and the pharmacological profile was found to be very similar to the human Y4 receptor. To study homo-and heterodimerization involving the Y4 receptor using bioluminescence resonance energy transfer 2 (BRET 2 ), the carboxy termini of the rhesus Y1, Y2, Y4, and Y5 receptors were fused to Renilla luciferase, and rhY4 was also fused to green fluorescent protein. Dimerization was also studied using Western blot analysis. Using both BRET 2 and Western analysis, we found that the rhY4 receptor is present at the cell surface as a homodimer. Furthermore, agonist stimulation using the Y4-selective agonists PP and 1229U91 can dissociate these dimers in a concentration-dependent manner. In contrast, rhY4 did not heterodimerize with other members of the NPY receptor family or with human opioid ␦ and receptors. Therefore, homodimerization is an important component in the regulation of the Y4 receptor.The neuropeptide Y (NPY) family of peptides consists of NPY, peptide YY (PYY), and pancreatic polypeptide (PP). These peptides regulate many important physiological functions, such as energy homeostasis, mood, and blood pressure. Currently, there are four cloned functional G-protein-coupled receptors (GPCRs), namely Y1, Y2, Y4, and Y5, that make up the NPY receptor family (see Berglund et al., 2003a for a review) in most mammals. All of these receptor subtypes are found in the brain as well as peripheral tissues. In addition, rabbits and mice have a functional y6 receptor, whereas in primates, this receptor subtype is not functional. NPY, PYY, and PP share a similar rank order of potencies at the Y1, Y2, and Y5 receptors: NPY Ϸ PYY Ͼ PP, whereas the Y4 receptor binds PP with higher affinity than it binds NPY and PYY and is thus regarded as the PP receptor. Like PP, the Y4 receptor is mainly found in the gut (Lundell et al., 1995), but binding sites for PP and Y4 mRNA have also been found in several rat brain regions, including hypothalamus and brainstem (Berglund et al., 2003a), suggesting that PP may also have direct effects on brain function. Besides the cloned NPY receptor family, several additional receptor subtypes have been proposed based on pharmacological evaluation of various tissue preparations. However, the first draft of the human genome did not provide evidence for any additional NPY receptor...

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“…Over 80% of NPY-containing neurons in the arcuate nucleus (Arc) coexpress Y2 receptor mRNA (9). This presynaptic Y2 receptor has therefore been proposed to regulate the release of NPY (10,11) and other colocalized neurotransmitters involved in energy homeostasis such as agouti related protein (AgRP) (12). The existence of such a feedback mechanism has been strengthened by recent reports that show that NPY release from rat hypothalamic slices is reduced by the Y2-preferring agonist NPY , and that this effect is blocked by the selective Y2 receptor antagonist (BIIE0246; ref.…”

mentioning

confidence: 99%

Important role of hypothalamic Y2 receptors in body weight regulation revealed in conditional knockout mice

Sainsbury

Schwarzer

Couzens

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

224

204

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Neuropeptide Y is implicated in energy homeostasis, and contributes to obesity when hypothalamic levels remain chronically elevated. To investigate the specific role of hypothalamic Y2 receptors in this process, we used a conditional Y2 knockout model, using the Cre-lox system and adenoviral delivery of Cre-recombinase. Hypothalamus-specific Y2-deleted mice showed a significant decrease in body weight and a significant increase in food intake that was associated with increased mRNA levels for the orexigenic NPY and AgRP, as well as the anorexic proopiomelanocortin (POMC) and cocaine-and amphetamine-regulated transcript (CART) in the arcuate nucleus. These hypothalamic changes persisted until at least 34 days after Y2 deletion, yet the effect on body weight and food intake subsided within this time. Plasma concentrations of pancreatic polypeptide and corticosterone were 3-to 5-fold increased in hypothalamus-specific Y2 knockout mice. Germ-line Y2 receptor knockout also produced a significant increase in plasma levels of pancreatic polypeptide. However, these mice differed from conditional knockout mice in that they showed a sustained reduction in body weight and adiposity associated with increased NPY and AgRP but decreased POMC and CART mRNA levels in the arcuate nucleus. The transience of the observed effects on food intake and body weight in the hypothalamus-specific Y2 knockout mice, and the difference of this model from germ-line Y2 knockout mice, underline the importance of conditional models of gene deletion, because developmental, secondary, or extrahypothalamic mechanisms may mask such effects in germ-line knockouts.neuropeptide Y ͉ pancreatic polypeptide ͉ cre-lox ͉ arcuate nucleus N europeptide Y (NPY) in the hypothalamus is known to be a strong stimulus for food intake (1, 2), and induces many neuroendocrine and metabolic changes that favor energy storage. Such changes include decreased thermogenesis in brown adipose tissue, hyperinsulinemia, hypercorticosteronemia, and insulin hyperresponsiveness in white adipose tissue (3, 4). All of these neuroendocrine and metabolic effects of central NPY administration persist even when NPY-induced hyperphagia is prevented by pair-feeding (3, 4), demonstrating that hyperphagia is not the only mechanism by which central NPY increases adiposity.Although numerous other hormones and peptides act within the hypothalamus to regulate energy homeostasis, many exert an important component of their effects via actions on the hypothalamic NPY-ergic system (5-7), demonstrating the pivotal role of NPY in coordinating energy homeostasis. However, it is not clear which of the 5 cloned Y-receptors (Y1, Y2, Y4, Y5, and y6) are responsible for these effects (8).There is increasing evidence that Y2 receptors are involved in energy homeostasis. Over 80% of NPY-containing neurons in the arcuate nucleus (Arc) coexpress Y2 receptor mRNA (9). This presynaptic Y2 receptor has therefore been proposed to regulate the release of NPY (10, 11) and other colocalized neurotransmitters involved ...

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Cloning and characterization of Rhesus monkey neuropeptide Y receptor subtypes

Cited by 19 publications

References 29 publications

The Use of Bioluminescence Resonance Energy Transfer 2 to Study Neuropeptide Y Receptor Agonist-Induced β-Arrestin 2 Interaction

The Use of Bioluminescence Resonance Energy Transfer 2 to Study Neuropeptide Y Receptor Agonist-Induced β-Arrestin 2 Interaction

Neuropeptide Y Y4 Receptor Homodimers Dissociate upon Agonist Stimulation

Important role of hypothalamic Y2 receptors in body weight regulation revealed in conditional knockout mice

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