G protein-linked receptors: Pharmacological evidence for the formation of heterodimers

By means of the expression of two chimeric receptors, alpha(2)/M-3 and M-3/ alpha(2), in which the carboxy-terminal receptor portions, containing trans membrane domains VI and VII, were exchanged between the alpha(2C)-adrenergi c and the M-3 muscarinic receptor, it has been shown that G protein-coupled receptors are able to interact functionally with each other at the molecul ar level to form (hetero) dimers. In the present study, we tested the hypot hesis that interaction between two different muscarinic receptor subtypes c an lead to the formation of a heterodimeric muscarinic receptor with a new pharmacological profile. Initially, muscarinic M-2 or M-3 wild-type recepto rs were expressed together with gene fragments originating from M-3 or M-2 receptors, respectively. Antagonist binding, performed with pirenzepine and tripitramine, revealed the presence of two populations of binding sites: o ne represents the wild-type M-2 or M-3 receptors, the other the heterodimer ic M-2/M-3 receptor. In another set of experiments, we constructed a point mutant M-2 receptor M-2 (Asn4043Ser), in which asparagine 404 was replaced by serine. Although this receptor alone did not show any binding for N-[H-3 ] methylscopolamine (up to 2 nM), when cotransfected with M-3, it resulted in the rescue of a high- affinity binding for tripitramine. These findings demonstrate that M-2 and M-3 muscarinic receptor subtypes can cross- intera ct with each other and form a new pharmacological heterodimeric receptor.