By means of the expression of two chimeric receptors, α2/m3 and m3/α2, in which the carboxyl-terminal receptor portions, containing transmembrane (TM) domains VI and VII, were exchanged between the α(2C) adrenergic and the m3 muscarinic receptor, Maggio et al. (Maggio, R., Vogel, Z., and Wess, J. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 3103-31073) demonstrated that G protein-linked 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 receptors might depend on the presence of a long third intracellular (i3) loop and that shortening this loop could impair the capability of receptors to form dimers. To address this question, we initially created short chimeric α2 adrenergic/m3 muscarinic receptors in which 196 amino acids were deleted from the i3 loop (α2/m3-short and m3/α2-short). Although co-transfection of α2/m3 and m3/α2 resulted in the appearance of specific binding, the co-expression of the two short constructs (α2/m3-short and m3/α2-short), either together or in combination, respectively, with m3/α2 and α2/m3 did not result in any detectable binding activity. In another set of experiments, a mutant m3 receptor, m3/m2(16aa), containing 16 amino acids of the m2 receptor sequence at the amino terminus of the third cytoplasmic loop, which was capable of binding muscarinic ligands but was virtually unable to stimulate phosphatidylinositol hydrolysis, was also mutated in the i3 loop, resulting in the m3/m2(16aa)-short receptor. Although co-transfection of m3/m2(16aa) with a truncated form of the m3 receptor (m3-trunc, containing an in frame stop codon after amino acid codon 272 of the rat m3 sequence) resulted in a considerable carbachol-stimulated phosphatidylinositol breakdown, the co-transfection of m3/m2(16aa)-short with the truncated form of the m3 receptor did not result in any recovery of the functional activity. Thus, these data suggest that intermolecular interaction between muscarinic receptors, involving the exchange of amino-terminal (containing TM domains I- V) and carboxyl-terminal (containing TM domains VI and VII) receptor fragments depends on the presence of a long i3 loop. One may speculate that when alternative forms of receptors with a different length of the i3 loop exist, they could have a different propensity to dimerize.
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