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 α2A-adrenergic receptor (AR) expression was driven by the endogenous mouse α2AAR gene locus. Exploiting this mouse line, we evaluated α2AAR trafficking and α2AAR-mediated inhibition of Ca2+ 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 α2AAR subtype. We discovered a more rapid desensitization of Ca2+ current suppression by clonidine than guanfacine, which paralleled a more marked receptor phosphorylation and endocytosis of α2AAR evoked by clonidine than by guanfacine. Clonidine-induced α 2AAR desensitization, but not receptor phosphorylation, was attenuated by blockade of endocytosis with concanavalin A, indicating a critical role for internalization of α2AAR 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-α2AAR 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. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.