Heterodimers of 1B- and 1D-Adrenergic Receptors Form a Single Functional Entity

Heterologous expression of 1D-adrenergic receptors ( 1DARs) in most cell types results in intracellular retention and little or no functionality. We showed previously that heterodimerization with 1B-ARs promotes surface localization of 1D-ARs. Here, we report that the 1B-/ 1D-AR interaction has significant effects on the pharmacology and signaling of the receptors, in addition to the effects on trafficking described previously. Upon coexpression of 1B-ARs and epitope-tagged 1D-ARs in both human embryonic kidney 293 and DDT1MF-2 cells, 1D-AR binding sites were not detectable with the 1D-AR selective antagonist 8-[2-(4-(2-methoxyphenyl)piperazin-1-yl)ethyl]-8azaspiro[4,5]decane-7,9-dione (BMY 7378), despite the ability to detect 1D-AR protein using confocal microscopy, immunoprecipitation, and a luminometer cell-surface assay. However, the 1B-AR-selective mutant F18A conotoxin showed a striking biphasic inhibition in 1B/ 1D-AR-expressing cells, revealing that 1D-ARs were expressed but did not bind BMY 7378 with high affinity. Studies of norepinephrine-stimulated inositol phosphate formation showed that maximal responses were greatest in 1B/ 1D-AR-coexpressing cells. Stable coexpression of an uncoupled mutant 1B-AR ( 12) with 1D-ARs resulted in increased responses to norepinephrine. However, Schild plots for inhibition of norepinephrine-stimulated inositol phosphate formation showed a single low-affinity site for BMY 7378. Thus, our findings suggest that 1B/ 1D-AR heterodimers form a single functional entity with enhanced functional activity relative to either subtype alone and a novel pharmacological profile. These data may help to explain why 1D-ARs are often pharmacologically undetectable in native tissues when they are coexpressed with 1B-ARs. An emerging paradigm in the field of pharmacology is that G-protein-coupled receptors (GPCRs) can form homoand heterodimers, resulting in the formation of unique multiprotein complexes that have altered trafficking, signaling, and pharmacological properties (Milligan et al., 2004; Terrillon and Bouvier, 2004; Prinster et al., 2005). In fact, recent data have raised the possibility that homodimerization may be a ubiquitous process that is required for the proper expression of GPCRs (Canals et al., 2004; Kaykas et al., 2004; Salahpour et al., 2004). A growing number of reports implicating a clinical role for GPCR dimerization in opiate analgesia (Jordan and Devi, 1999), human immunodeficiency virus infection (Rodriguez-Frade et al., 2004), and vitreoretinopathy (Kaykas et al., 2004) highlight the need to continue characterizing the mechanisms and properties of novel GPCR dimers. Numerous studies have now shown that GPCR heterodimerization is essential for proper expression and function of GABAB (Marshall et al., 1999), taste (Nelson et al., 2001), olfactory (Hague et al., 2004b), and 1D-adrenergic receptors (ARs) (Hague et al., 2004c). The most convincing and thoroughly studied example to date of GPCR heterodimerization involves the formation of functional GABAB receptors. It is now clear that GABABR1 and GABABR2 must heterodimerize to ensure trafficking of GABAB receptors to the cell surface (Kaupmann et al., 1998; Marshall et al., 1999) at least partially through the masking of an endoplasmic reticulum (ER) retention signal located in the carboxylterminal tail of GABABR1 receptors (Margeta-Mitrovic et al., 2000). In addition, the formation of sweet taste receptors requires heterodimerization of T1R2 and T1R3 receptors (Nelson et al., 2001), and the M71 mouse olfactory receptor can achieve surface expression and become functional when heterodimerized with the 2-AR (Hague et al., 2004b). In previous studies, we showed that 1D-AR heterodimerization Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.105.014985. ABBREVIATIONS: GPCR, G-protein-coupled receptor; AR, adrenergic receptor; NE, norepinephrine; InsP, inositol phosphate; GFP, green fluorescent protein; HEK, human embryonic kidney; HA, hemagglutinin; ER, endoplasmic reticulum; WT, wild type; PI, phosphatidylinositol; 5-MU, 5-methylurapidil; PBS, phosphate-buffered saline; BE 2254, 2(4-hydroxyphenyl)-ethylaminomethyl)-tetralone; BMY 7378, 8-[2-(4-(2methoxyphenyl)piperazin-1-yl)ethyl]-8-azaspiro[4,5]decane-7,9-dione. 0026-895X/06/6901-45–55$20.00 MOLECULAR PHARMACOLOGY Vol. 69, No. 1 Copyright © 2006 The American Society for Pharmacology and Experimental Therapeutics 14985/3069012 Mol Pharmacol 69:45–55, 2006 Printed in U.S.A.