The C-terminal half of the α2C-adrenoceptor determines the receptor's membrane expression level and drug selectivity

Abstract

In tissues as well as in transfected cells, α2C-adrenoceptors show poorer expression levels compared to α2A-adrenoceptors. In order to characterize which regions of the α2C-adrenoceptor are involved in regulating the expression of binding-competent receptors at the plasma membrane, six chimeric α2A-/α2C-adrenoceptors were constructed. The wild-type α2A- and α2C-adrenoceptors and the six chimeric α2A-/α2C-adrenoceptors were transiently transfected into human embryonic kidney 293 (HEK293) cells, and the expression levels were investigated by radioligand binding. The results show that the C-terminal half of the α2C-adrenoceptor, ranging from the second extracellular loop to the C-terminus, is the main determinant of the low expression level of binding-competent α2C-adrenoceptors in HEK293 cell membranes. The so-called retention signal in the N-terminus of the α2C-adrenoceptor had a less profound effect on the expression levels of the chimeric receptors. For seven drugs competing for [(3)H]-RX821002 binding, the K i values were determined at the wild-type α2A- and α2C-adrenoceptors and at four of the chimeric α2A-/α2C-adrenoceptors. The results show that the α2C- over α2A-selectivity of spiroxatrine, spiperone, clozapine, MK912, and chlorpromazine, as well as the α2A- over α2C-selectivity of BRL44408, resides mainly in the C-terminal half of the receptors. To some extent, the region comprising the N-terminal half of the receptors contributed to the α2C-selectivity of spiperone, clozapine, and chlorpromazine.

Expression and trafficking of functional G protein-coupled receptors are related, yet distinct, concepts

“…Delineation of amino acid motifs in GPCRs that modulate PM expression or trafficking often requires creation of chimeric or mutant receptors, followed by cellular analysis of receptor levels by either ligand binding or antibody-based methods (Angelotti et al 2010;Laurila et al 2011;Jahnsen and Uhlen 2013). Investigators must be cognizant that ligand bindingcompetent receptor folding and transport-competent receptor folding can be altered inadvertently by construction of chimeric/mutant GPCRs.…”