2C-Adrenergic Receptors Exhibit Enhanced Surface Expression and Signaling upon Association with 2-Adrenergic Receptors

The 2C-adrenergic receptor ( 2CAR) is known to be poorly trafficked to the cell surface when expressed in a variety of cell types. We tested the hypothesis that the surface expression and signaling of 2CAR might be enhanced by heterodimerization with other G protein-coupled receptors (GPCRs). Cotransfection of 2CAR with more than 25 related GPCRs revealed that only coexpression with the 2-adrenergic receptor ( 2AR) increased the surface localization of 2CAR in human embryonic kidney-293 cells. Coimmunoprecipitation of 2CAR with 2AR confirmed a physical interaction between the two receptors. Confocal microscopy studies demonstrated that 2CAR expressed alone was mainly intracellular, whereas 2CAR coexpressed with 2AR was predominantly localized to the plasma membrane. Ligand binding studies revealed a significant increase in 2CAR binding sites upon coexpression with 2AR, with no apparent change in affinity for 2AR ligands. Functional assays with the 2AR-specific agonist brimonidine (UK 14,304) revealed that coexpression of 2AR with 2CAR enhanced 2CAR-mediated activation of extracellular signalregulated kinase 1/2. Furthermore, analyses of agonist-promoted receptor endocytosis demonstrated enhanced 2CAR internalization in response to 2AR agonists when 2CAR and 2AR were coexpressed. In addition, substantial cointernalization of 2CAR in response to AR agonists was observed when 2CAR was coexpressed with 2AR. These data reveal that 2CAR can interact with 2AR in cells in a manner that regulates 2CAR surface expression, internalization, and functionality. The adrenergic receptors are a family of cell-surface G protein-coupled receptors (GPCRs) that mediate the actions of the hormone epinephrine and the neurotransmitter norepinephrine. The three main adrenergic receptor (AR) classes ( 1, 2, and 2) can be further divided into three subtypes each, and all of these subtypes are excellent targets for therapeutic pharmaceuticals. The specific roles of the various adrenergic receptor subtypes is becoming increasingly clear through studies on knock-out mice (Philipp and Hein, 2004), and novel therapies making use of these insights await the development of more subtype-specific drugs. However, two of the adrenergic receptor subtypes, 2CAR and 1DAR, have proven extremely difficult to study in heterologous expression systems, because they do not traffic efficiently to the cell surface when expressed alone and are therefore largely nonfunctional (von Zastrow et al., 1993; Daunt et al., 1997; Chalothorn et al., 2002). Recently, it has been shown that 1DAR surface expression and functionality can be profoundly enhanced by coexpression with 1BAR or 2AR, presumably due to receptor heterodimerization (Uberti et al., 2003, 2005; Hague et al., 2004b). The mechanisms underlying the 2CAR-trafficking defect remain enigmatic and are important to address because of the therapeutic importance of drugs targeting 2 receptors. It has been shown that 2CAR does traffic efficiently to the cell surface when expressed in several neuronally derived cell types, suggesting that the poor trafficking of 2CAR seen in other cell types is highly dependent on cellular context (Hurt et al., 2000). Other studies suggest that surface expression of 2CAR can be increased by exposure to cold temperatures, which may further contribute to tissue-specific regulation of 2CAR activity (Jeyaraj et al., 2001; Bailey et al., 2004). Studies on 2CAR knockout mice reveal a key role for this subtype in mediating spinal analgesia (Fairbanks et al., 2002) and in the regulation of epinephrine release (Hein et This work was supported by grants from the National Institutes of Health and W. M. Keck Foundation. 1 Current affiliation: Department of Pharmaceutical Sciences, Nesbitt School of Pharmacy, Wilkes University, Wilkes-Barre, Pennsylvania. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.106.106526. ABBREVIATIONS: GPCR, G protein-coupled receptor; AR, adrenergic receptor; GABABR, GABAB receptor; DHA, dihydroalprenolol; ECL, enhanced chemiluminescence; ELISA, enzyme-linked immunosorbent assay; ERK, extracellular signal-regulated kinase; HA, hemagglutinin; HEK, human embryonic kidney; PAGE, polyacrylamide gel electrophoresis; RX 821002, 2-methoxyidazoxan; UK 14,304, brimonidine. 0022-3565/06/3183-974–981$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 318, No. 3 Copyright © 2006 by The American Society for Pharmacology and Experimental Therapeutics 106526/3132675 JPET 318:974–981, 2006 Printed in U.S.A.