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 transmembrane domains VI and VII, were exchanged between the alpha(2C)-adrenergic 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 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 M(2) or M(3) wild-type receptors 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: one represents the wild-type M(2) or M(3) receptors, the other the heterodimeric M(2)/M(3) receptor. In another set of experiments, we constructed a point mutant M(2) receptor M(2) (Asn404-->Ser), in which asparagine 404 was replaced by serine. Although this receptor alone did not show any binding for N-[(3)H]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-interact with each other and form a new pharmacological heterodimeric receptor.

Antagonist binding profile of the split chimeric muscarinic m2-trunc/m3-tail receptor.

Recent evidence suggests that G-protein-coupled receptors can behave as multiple subunit receptors, and can be split into parts, maintaining their binding ability. Transfection of a truncated muscarinic m2 receptor (containing transmembrane domains I-V, named m2-trunc) with a gene fragment coding for the carboxyl-terminal receptor portion of the muscarinic m3 receptor (containing transmembrane domains VI and VII, named m3-tail) results in the formation of a binding site with a high affinity for the muscarinic ligand N-[3H]methylscopolamine. In this paper we analyse the antagonist binding profile of this chimeric m2-trunc/m3-tail receptor in comparison with the wild-type muscarinic m2 and m3 receptors. While many of the substances tested had an intermediate affinity for the chimeric m2-trunc/m3-tail receptor compared with m2 and m3, some compounds were able to distinguish between the chimeric m2-trunc/m3-tail receptor on the one hand and the m2 or the m3 receptor on the other. Among them, tripitramine (a high-affinity M2 receptor antagonist) bound to the m2-trunc/m3-tail receptor with the same affinity as m2, but it bound to the m3 receptor with a 103-fold lower affinity; pirenzepine (a selective muscarinic M1 receptor antagonist) bound to the chimeric receptor with an affinity that was 12- and 3-fold higher than that of m2 and m3, respectively. The results of this study demonstrate that the chimeric m2-trunc/m3-tail receptor has a pharmacological profile distinct from that of the originating muscarinic m2 and m3 receptors.

Pergolide binds tightly to dopamine D2 short receptors and induces receptor sequestration.

Pergolide is an ergotamine derivative with potent D1 and D2 receptor activity. In this study we showed that pergolide binds tightly to dopamine D2 short receptors, as indicated by the long period of occupancy of the receptors after washing. Furthermore, pergolide induces receptor internalization to a larger extent than dopamine, seeing that no recycling of the receptors to the plasma membrane was observed for either agonist. The dissociation of pergolide from dopamine receptors occurs during the endocytotic process, leaving the receptors accessible to [3H]methylspiperone. Pergolide is a lipophilic compound that can reach and compete with [3H]methylspiperone for binding to sequestered receptors. If internalized receptors are still a target for drug action, pergolide could be a suitable compound of therapeutic interest in cases where receptor sequestration could prevent dopamine efficacy, as in levodopa therapy.