Oligomerization of opioid receptors Ivone Gomes, Julija Filipovska, Bryen A. Jordan, 1 and Lakshmi A. Devi * Department of Pharmacology, New York University School of Medicine, MSB 408, 550 First Avenue, New York, NY 10016, USA Accepted 4 June 2002 Abstract Opioid receptors belong to the family of G-protein-coupled receptors characterized by their seven transmembrane domains. The activation of these receptors by agonists such as morphine and endogenous opioid peptides leads to the activation of inhibitory G- proteins followed by a decrease in the levels of intracellular cAMP. Opioid receptor activation is also associated with the opening of K þ channels and the inhibition of Ca 2þ channels. A number of investigations, prior to the development of opioid receptor cDNAs, suggested that opioid receptor types interacted with each other. Early pharmacological studies provided evidence for the probable interaction between opioid receptors. More recent studies using receptor selective antagonists, antisense oligonucleotides, or animals lacking opioid receptors further suggested that interactions between opioid receptor types could modulate their activity. We ex- amined opioid receptor interactions using biochemical, biophysical, and pharmacological techniques. We used differential epitope taggingandselectiveimmunoisolationofreceptorcomplexestodemonstratehomotypicandheterotypicinteractionsbetweenopioid receptor types. We also used the proximity-based bioluminescence resonance energy transfer assay to explore opioid receptor–re- ceptor interactions in living cells. In this article we describe the biochemical and biophysical methods involved in the detection of receptor dimers. We also address some of the concerns and suggest precautions to be taken in studies examining receptor–receptor interactions. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Immunoprecipitation; Western blotting; Crosslinking; Bioluminescence resonance energy transfer 1. Biochemical techniques to study opioid receptor oligo- merization To demonstrate receptor–receptor interactions using biochemical methods different epitope-tagged receptors (Flag- and myc-taggedversionsofthesamereceptor)are expressed in heterologous cells. After membrane solu- bilization myc-tagged receptors are immunoprecipitated using anti-myc polyclonal antibodies; the immunopre- cipitates are subjected to separation by SDS–poly- acrylamide gel electrophoresis (SDS–PAGE). The associated receptors in the complex are visualized by Western blot with anti-Flag monoclonal antibodies. A signal is detected in the blots only if there is an associ- ation between the myc- and Flag-tagged receptors. 1.1. Experimental protocols 1.1.1. Transient transfection and crosslinking We have typically used opioid receptors that have been tagged at the N termini with Flag, myc, or HA epitope tags. Human embryonic kidney 293 (HEK-293) or COS cells are used for the transient expression of these receptors since these cells are easy to grow, easy to transfect, express heterologous proteins at fairly high levels, and do not normally express opioid receptors. To transiently express these receptors we routinely use cal- cium phosphate-mediated transfection. In this method a calcium phosphate–DNA precipitate is formed by slowly mixing Hepes-buffered saline with a solution containing calcium chloride and the DNA to be trans- fected. This precipitate adheres to the surface of the cells to be transfected and ‘‘glycerol shock’’ increases the amount of DNA absorbed by the cells. HEK-293 or COS cells (American Type Culture Collection, Manassas, VA) are grown in the growth medium[Dulbecco’smodifiedEagle’smedium(DMEM, Methods 27 (2002) 358–365 www.academicpress.com * Corresponding author. Fax: +212-263-7133. E-mail address: lakshmi.devi@med.nyu.edu (L.A. Devi). 1 Present address: Department of Biochemistry, NYU School of Medicine, 550 First Avenue, New York, NY 10016, USA. 1046-2023/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII:S1046-2023(02)00094-4