Mammalian NPY Receptor Subtypes

The diverse biological effects of NPY are mediated by the activation of different receptor subtypes which are all members of the larger superfamily of G-protein coupled receptors (GPCRs). In mammals five neuropeptide Y receptor subtypes have been described, denoted as Y1, Y2, Y4, Y5, and y6.^41-48 The y6 receptor was found to be functional in mice, but is a non-functional pseudogene in most mammalian species. In the rat genome it is missing at all.⁴⁹ In Table 1.1 an overview of the binding profiles, the localization and the physiological role of the mammalian NPY receptor subtypes is given.

Table 1.1. Overview of binding profile, localization and physiological role of the mammalian NPY receptor subtypes.^{27, 50-53}

Binding Profile Localization Physiological Role

Y1

> NPY13-36 > rPP hypothalamus food intake, seizures, anxiety

muscles no function in humans

1.2.1 The Y1, Y4 and Y5 Receptor

The 384 amino acids containing Y1R was the first NPY receptor to be cloned in 1992.

The functionality of the expressed Y1R was demonstrated by inhibition of adenylyl cyclase (AC) and mobilization of intracellular calcium. All mammalian Y1Rs cloned thus far display 90-95 % homology with the human Y1R (hY1R).⁵⁵ The Y1R exhibits a high tendency to agonist stimulated internalization, as demonstrated by means of radioligand binding,⁵⁶ confocal microscopy⁵⁷ and GFP tagged Y1R.⁵⁸ Interestingly, the pseudopeptide Y1R antagonist GR231118 is able to induce long-lasting disappearance of cell surface Y1Rs through a mechanism distinct from the classical endocytot-ic/recycling pathway.⁵⁹

Sequence homology screening using a Y1R probe led to the isolation of a new human NPY receptor cDNA, encoding for the Y4 receptor (Y4R).^{47, 60-61} A unique feature of the Y4R is a high affinity for PP, in contrast to NPY and PYY, which exhibit low affinities for this receptor. Therefore, the Y4R is also referred to as PP receptor. Sequence homology between human and other species Y4Rs is one of the lowest (less than 75 %) reported for orthologous GPCRs of different mammalian species.⁴⁹ Additionally, the human Y4R (hY4R) has higher homology with hY1R (43 %) than human Y2 receptor (hY2R; 34 %).⁶² Moreover, there are contrary observations in terms of receptor inter-nalization.^{56, 63}

NPY is one of the most potent orexigenic agents and this effect was proposed to be mediated by an atypical Y1R or “feeding receptor”.^64-65 The pharmacological profile of an NPY receptor cloned from human and rat tissues was found to be similar to that of this atypical feeding receptor and classified as the Y5 receptor subtype (Y5R).^{41, 45} In comparison with other NPY receptor subtypes, this 446 amino acid protein has a very long third intracellular loop with more than 130 amino acids and an unusually short C-terminus. Additionally, sequence homology between the Y5R and other NPY receptors is very low (≈ 30 %).^{49, 55} Lastly, rapid association of β-arrestin 2 with the Y5R was observed after agonist stimulation, suggesting fast internalization similar to the Y1R.⁶⁶

1.2.2 The NPY Y2 Receptor

In 1986, the Y2 subtype was pharmacologically characterized as a predominantly presynaptic NPY/PYY receptor.⁴ The cloned hY2R consists of 381 amino acids and has only ≈ 30 % identity to the Y1R and the Y4R, respectively.42, 48, 61, 68-69

However, high sequence homology (90-95 %) between species was observed for various Y2Rs.^{49, 55} Activation of the Y2R leads to an inhibition of AC⁷⁰ and in rat neurons to an inhibition of Ca²⁺ influx via N-type channels^71-72. The human glioblastoma cell line LN319 proved useful in defining Y2-mediated pathways, since these cells exclusively express this NPY receptor subtype.⁷³ Herein, NPY inhibits

forskolin-induced cAMP accumulation and stimulates an increase in intracellular Ca²⁺ via pertussis toxin-sensitive pathways.⁷⁴ Further-more, the Y2R mediates the activation of protein kinase C (PKC) in human ciliated cells.⁷⁵ Finally, the Y2R was also shown to activate MAPK signal transduction through a Gi protein and via PI-3 kinase.⁷⁶ The different signaling pathways are summarized in Figure 1.4.

The Y2R internalization process was a matter of controversy over the past years. Desensitiza-tion of the Y2R after stimulation with NPY was observed in LN319 cells,⁷⁴ but unlike the Y1R, the Y2R has not been reported to internalize after prolonged agonist excitation^{56, 58} or it was reported to internalize very slowly.⁷³ However,

mutagenesis studies recently revealed that substitution of either His155 or His159 by Pro in the intracellular loop 2 (ICL2) can lead to an accelerated internalization.⁷⁷ Notably, the postulated regulation of the Y2R internalization by its ICL2 is inconsistent with the most recent findings on Y1/Y2R chimeras, revealing putative inhibitory interactions within the ICL3 and the C-terminal tail of the native Y2R that reduce internalization.⁷⁸ In the meantime, several groups confirmed rapid Y2R inter-nalization.^79-82

1.2.3 The Y2R in Health and Disease

The high sequence homology of Y2Rs in mammals reflects the involvement of the Y2R in critical developmental and metabolic events. Thus, the Y2R is also involved in a variety of human diseases such as epilepsy, obesity and cancer. Especially, the role of the NPY receptor family in appetite regulation has received intense attention in recent years,⁸³ as obesity has emerged as one of the most serious major human health concerns in the present and future⁸⁴. Herein, the Y2R appeared as interesting

Figure 1.4. The neuropeptide Y Y2 receptor and its intracellular signaling pathways, adapted from Brumovsky et al.⁶⁷

target as numerous studies reported reduced hunger and food intake in humans after application of the moderately Y2R selective agonist PYY(3-36).^85-86 The potential benefits of Y2 and Y4R agonism for the treatment of obesity resulted in Y2/Y4 dual agonists developed by 7TM Pharma. The most promising peptide was tested successfully in Phase I/II trials and is currently under further investigation proving the potential of the Y2R as anti-obesity drug target.³⁷ Furthermore, Y2R agonism was observed to be neuroprotective, presumably, via the reduction of glutamate release.

Thus, the Y2R is discussed as a potential drug target in seizures and epilepsy.⁸⁷ Moreover, wound healing is reduced in Y2 knock-out mice.⁸⁸ Further functions of the Y2R which are related to pathological processes were recently summarized.⁶⁹

Besides its physiological implications and potential role in diverse dysregulated physiological processes, the Y2R attracted strong attendance on its involvement in oncogenesis and has recently been predicted as tumor marker.⁸⁹ A remarkably high expression was found in various human brain tumors and mastocarcinoma.⁹⁰ Blocking the Y2Rs led to an inhibition of neuroblastoma growth in vivo, emphasizing the possible application of selective and potent Y2R antagonists in cancer therapy.⁹¹ Recently, a carbaborane modified NPY analog was prepared for potential application in cancer treatment (boron neutron capture therapy).⁹² Thus, the Y2R is considered to be a promising target in tumor therapy and imaging, respectively.