D-2/D-3 dopamine receptor heterodimers exhibit unique functional properties

Evidence for heterodimerization has recently been provided for dopamine D-1 and adenosine A(1) receptors as well as for dopamine D-2 and somatostatin SSTR5 receptors. In this paper, we have studied the possibility that D2 and Da receptors interact functionally by forming receptor heterodimers. Initi ally, we split the two receptors at the level of the third cytoplasmic loop into two fragments. The first, containing transmembrane domains (TM) I to V and the N-terminal part of the third cytoplasmic loop, was named D-2trunk or D-3trunk, and the second, containing the C-terminal part of the third c ytoplasmic loop, TAM and TMVII, and the C-terminal tail, was named D-2tail or D-3tail. Then we defined the pharmacological profiles of the homologous (D-2trunk/ D-2tail and D-3trunk/D-3tail) as well as of the heterologous (D- 2trunk/D-3tail and D-3trunk/D-2tail) cotransfected receptor fragments. The pharmacological profile of the cross-cotransfected fragments was different from that of the native D-2 or D-3 receptors. In most cases, the D-3trunk/D -2tail was the one with the highest affinity for most agonists and antagoni sts. Moreover, we observed that all of these receptor fragments reduced the expression of the wild type dopamine D-2, and D-3 receptors, suggesting th at D-2 and D-3 receptors can form complexes with these fragments and that t hese complexes bind [H-3]nemonapride less efficiently or are not correctly targeted to the membrane. In a second set of experiments, we tested the abi lity of the split and the wild type receptors to inhibit adenylyl cyclase ( AC) types V and VI. All of the native and split receptors inhibited AC-V an d AC-VI, with the exception of D-3, which was unable to inhibit AC-VI. We t herefore studied the ability of D2 and D3 to interact functionally with one another to inhibit AC-VI. We found that with D-2 alone, R-(+)-7-hydroxydyp ropylaminotetralin hydrobromide inhibited AC-VI with an IC50 of 2.05 +/- 0. 15 nM, while in the presence of D2 and D3 it inhibited AC-Vr with an IC50 o f 0.083 +/- 0.011 nM. Similar results were obtained with a chimeric cyclase made from AC-V and AC-VI. Coimmunoprecipitation experiments indicate that D-2 and D-3 receptors are capable of physical interaction.