G protein-linked receptors/ pharmacological evidence for the formation of heterodimers

By means of the expression of two chimeric receptors, α2/M3 and M3/α2, in which the carboxy-terminal receptor portions, containing transmembrane domains VI and VII, were exchanged between the α(2C)- adrenergic and the M3 muscarinic receptor, it has been shown that G protein- coupled receptors are able to interact functionally with each other at the molecular level to form (hetero)dimers. In the present study, we tested the hypothesis that interaction between two different muscarinic receptor subtypes can lead to the formation of a heterodimeric muscarinic receptor with a new pharmacological profile. Initially, muscarinic M2 or M3 wild- type receptors were expressed together with gene fragments originating from M3 or M2 receptors, respectively. Antagonist binding, performed with pirenzepine and tripitramine, revealed the presence of two populations of binding sites: one represents the wild-type M2 or M3 receptors, the other the heterodimeric M2/M3 receptor. In another set of experiments, we constructed a point mutant M2 receptor M2 (Asn404→Ser), in which asparagine 404 was replaced by serine. Although this receptor alone did not show any binding for N-[3H]methylscopolamine (up to 2 nM), when cotransfected with M3, it resulted in the rescue of a high-affinity binding for tripitramine. These findings demonstrate that M2 and M3 muscarinic receptor subtypes can cross-interact with each other and form a new pharmacological heterodimeric receptor.