Abstract
Although ligand-selective regulation of G protein-coupled receptor-mediated signaling and trafficking are well documented, little is known about whether ligand-selective effects occur on endogenous receptors or whether such effects modify the signaling response in physiologically relevant cells. Using a gene targeting approach, we generated a knock-in mouse line, in which N-terminal hemagglutinin epitope-tagged alpha(2A)-adrenergic receptor (AR) expression was driven by the endogenous mouse alpha(2A)AR gene locus. Exploiting this mouse line, we evaluated alpha(2A)AR trafficking and alpha(2A)AR-mediated inhibition of Ca(2+) currents in native sympathetic neurons in response to clonidine and guanfacine, two drugs used for treatment of hypertension, attention deficit and hyperactivity disorder, and enhancement of analgesia through actions on the alpha(2A)AR subtype. We discovered a more rapid desensitization of Ca(2+) current suppression by clonidine than guanfacine, which paralleled a more marked receptor phosphorylation and endocytosis of alpha(2A)AR evoked by clonidine than by guanfacine. Clonidine-induced alpha(2A)AR desensitization, but not receptor phosphorylation, was attenuated by blockade of endocytosis with concanavalin A, indicating a critical role for internalization of alpha(2A)AR in desensitization to this ligand. Our data on endogenous receptor-mediated signaling and trafficking in native cells reveal not only differential regulation of G protein-coupled receptor endocytosis by different ligands, but also a differential contribution of receptor endocytosis to signaling desensitization. Taken together, our data suggest that these HA-alpha(2A)AR knock-in mice will serve as an important model in developing ligands to favor endocytosis or nonendocytosis of receptors, depending on the target cell and pathophysiology being addressed.
Highlights
G protein-coupled receptors (GPCRs)4 represent the largest family of cell surface receptors mediating responses to hormones, cytokines, neurotransmitters, and therapeutic agents [1]
Binding of arrestins to GPCRs leads to GPCR internalization [4, 5], a process that has been proposed as a means to desensitize receptor signaling at the cell surface, resensitize receptors, and/or initiate intracellular signaling [6, 7]
Blockade of Internalization Attenuates Desensitization of the data reveal that N-terminal HA-tagged ␣2AAR in a knock-in Clonidine-induced Response but Has No Effect on Desensitiza- mouse fully mimics the WT receptor in terms of ␣2AAR distrition of the Guanfacine-induced Response—we sought to bution (Fig. 2), receptor density, intrinsic receptor binding directly address the causal relationship between greater affinity, and G protein activation (Fig. 1)
Summary
G protein-coupled receptors (GPCRs)4 represent the largest family of cell surface receptors mediating responses to hormones, cytokines, neurotransmitters, and therapeutic agents [1]. Clonidine Evokes a Sustained and Greater Desensitization of Ca2ϩ Current Inhibition than Guanfacine in Cultured SCG Neurons—Because both receptor phosphorylation and internalization have been proposed as major mechanisms in regulating signaling response duration evoked by the receptor [2, 6], we postulated that the observed differential abilities of clonidine and guanfacine to promote ␣2AAR phosphorylation and internalization might result in different desensitization profiles of ␣2AAR-mediated signaling processes in response to these two drugs.
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
