Construction of covalently coupled, concatameric dimers of 7TM receptors

Abstract

7TM receptors are easily fused to proteins such as G proteins and arrestin but because of the fact that their terminals are found on each side of the membrane they cannot be joined directly in covalent dimers. Here, we use an artificial connector comprising a transmembrane helix composed of Leu-Ala repeats flanked by flexible spacers and positively charged residues to ensure correct inside-out orientation plus an extracellular HA-tag to construct covalently coupled dimers of 7TM receptors. Such 15 TM concatameric homo- and heterodimers of the β₂-adrenergic and the NK₁ receptors, which normally do not dimerize with each other, were expressed surprisingly well at the cell surface, where they bound ligands and activated signal transduction in a manner rather similar to the corresponding wild-type receptors. The concatameric heterodimers internalized upon stimulation with agonists for either of the protomers, which was not observed upon simple coexpression of the two receptors. It is concluded that covalently joined 7TM receptor dimers with surprisingly normal receptor properties can be constructed with use of an artificial transmembrane connector, which perhaps can be used to fuse other membrane proteins.

Keywords::

• 7TM receptors
• dimerization
• β2-adrenergic receptor
• neurokinin receptor type 1
• concatamers

Acknowledgments

This study was supported by grants from the Danish Medical Research Council and from the European Community’s Sixth Framework Programme (grant LSHB-CT-2003-503337). M. Terpager was recipient of a Ph.D. scholarship from the Faculty of Health Sciences, University of Copenhagen. V. Kubale was supported by the Slovenian Research Agency with Slovenian-Danish collaboration grant (BI-DK/06-07-007). We thank Drs. Maria Waldhör and Bo van Deurs for advice and support in relation to the immunofluorescence microscopy studies, Prof. Milka Vrecl for proofreading manuscript and Mette Simons for expert technical assistance.

Declaration of interest: The authors declare no conflict of interest.