Co-expression of neuropeptide Y Y1 and Y5 receptors results in heterodimerization and altered functional properties
Introduction
Peptide YY (PYY), pancreatic polypeptide (PP) and neuropeptide Y (NPY) constitute a family of endocrine and neuronally derived peptides that have important physiological functions [1]. NPY is found in central and peripheral neurons and is released into the circulation from the adrenals. The effects of these peptides are mediated through G-protein-coupled receptors (class 1) entitled Y1, Y2, Y4 and Y5 (for review see [2]). An additional receptor, y6 has been identified in mice but is not functional in rats and humans [3]. The endogenous ligand for the Y4 receptor appears to be PP while Y1, Y2 and Y5 have similar high affinities for NPY and PYY [2]. Centrally administered NPY and PYY produce a pronounced orexigenic effect, anxiolysis, antiepileptic action, altered hormonal responses, altered diurnal functions and cardiovascular responses. As such, this system has been an important target for further research and pharmaceutical discovery.
A number of studies have been conducted to understand the pharmacological specificity of the central responses to NPY and PYY. In particular, the receptor mediating the orexigenic responses has been pursued by a number of groups. Early studies using peptide analogs indicated an Y1-like pharmacological profile, however, the peptide responses were “atypical”[4], [5]. The robust activity of the fragment NPY2-36 was particularly puzzling since it had greater efficacy in stimulating the feeding responses than NPY but lower affinity for the Y1 receptor in vitro. Subsequent studies using more specific peptide analogs and antagonists indicated that both Y1 and Y5 receptors could mediate the feeding response. When NPY and PYY are administered to mice with the Y1 or Y5 receptor genes deleted, a partial reduction of the feeding response was observed (for review see [6]. However, these studies are confounded by the expression of a functional y6 receptor in mice that may be involved in the feeding responses observed in this species [3], [7]. A similar profile has been observed in anxiety studies where both Y1 and Y5 receptor-selective agonists can produce anxiolytic-like activities after central administration [8]. Taken as a whole, these studies suggest that Y1 and Y5 receptors mediate similar actions in the brain and their combined pharmacologies may account for the “atypical Y1 receptor-like” pharmacology observed in earlier studies.
Since Y1 and Y5 receptor agonists produce similar physiological properties after central administration, it would be logical that they would be found in similar brain regions. The Y1 and Y5 receptor genes are found in close proximity but opposite and overlapping orientation on chromosome 11, suggesting coordinate regulation [9]. In the rat brain, Y5 receptor mRNA expression was found to coincide with regions expressing Y1 receptor mRNA [10]. Furthermore, using double label immunohistofluorescence, Wolak et al. [11] demonstrated that the Y1 and Y5 receptor proteins were colocalized in many brain regions and, in many instances, to the same cells. Since both receptors mediate their functions through Gi, it would be interesting to know if co-expression of these receptors in the same cell line would result in enhanced function or altered pharmacological properties. Recent work with a variety G-protein coupled receptors has proven that co-expression can result in heterodimerization [12], [13] leading to alterations in pharmacology, desensitization and functional responses [14], [15].
A number of techniques have been used to demonstrate G-protein-coupled receptor dimerization (or oligomerization) [16]. Resonance energy transfer methods provide evidence that the receptors are in close proximity, allow for assessment of agonist and antagonist interactions and can be performed in living cells. Therefore, we tested the hypothesis that co-expression leads to a heterodimerization of Y1 and Y5 receptors using the well-established technique of bioluminescence resonance energy transfer (BRET). To accomplish these goals, Renilla luciferase (RLUC) and green fluorescent protein (GFP) were attached to the C-terminus of the Y1 and Y5 receptor subtypes, respectively, and the receptor proximity assessed using a BRET assay. Receptor functionality was assessed using binding and functional adenylyl cyclase assays. Receptor internalization was assessed using confocal microscopy.
Section snippets
Receptor expression construct assembly
The cloning of the Y1 and Y5 receptors from the rhesus monkey was reported previously [17]. Using clones containing the coding sequence of the Y1 and Y5 receptors, one forward primer was synthesized for each: Y1.fH containing a HindIII site with the sequence: (5′-AAGCTTAAGCTTACCATGAATTCAACATTATTTTCCC AG-3′) and Y5.fH (5′-AAGCTTAAGCTTACCATGGATTTAGAGCTCGATGA AT-3′). One reverse primer was also synthesized for each Y1.NSrKS (5′-CCGCGGTACCGAT TCTTTCATTATCATCATTGTTG-3′), and Y5.NSrKS
Radioligand-binding analysis of wild-type versus tagged receptors
These initial experiments were performed to determine if the addition of the GFP and RLUC tags affected the ligand recognition properties of the Y1 and Y5 receptors in radioligand binding assays (Supplemental Figures 1–7). In these studies, 125I-PYY was found to bind with similar affinity to the wild-type and tagged receptors (Supplemental Figure 1) with specific binding constituting greater than 80% of total binding. Subsequently, the pharmacology of [125I]-PYY binding to the cell lines was
Discussion
In the present study, we have demonstrated that the Y1 and Y5 receptors exhibit profound interactions when co-expressed. When expressed at similar densities as described in Section 3, tagged Y1 and Y5 receptors exhibit an increased BRET ratio compared to other Y receptor pairings. The endogenous agonists, NPY and PYY, had no effect on the BRET ratio and exhibited similar levels of potency when comparing the Y5 cell line with the Y1/Y5 cell line in binding assays. On the other hand, Y5 agonists
Acknowledgement
This work was supported by Eli Lilly and Company.
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Present address: Preclinical Development, Biovitrum AB, SE-11276 Stockholm, Sweden.