FUNCTIONAL-ROLE OF THE 3RD CYTOPLASMIC LOOP IN MUSCARINIC RECEPTOR DIMERIZATION

By means of the expression of two chimeric receptors, alpha(2)/m3 and m3/alpha(2), in which the carboxyl-terminal receptor portions, contain ing transmembrane (TM) domains VI and VII, were exchanged between the alpha(2C) adrenergic and the m3 muscarinic receptor, Maggio ct al, (Ma ggio, 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 fo rm (hetero)dimers. In the present study we tested the hypothesis that interaction between receptors might depend on the presence of a long t hird intracellular (i3) loop and that shortening this loop could impai r the capability of receptors to form dimers, To address this question , we initially created short chimeric alpha(2) adrenergic/m3 muscarini c receptors in which 196 amino acids were deleted from the i3 loop (al pha(2)/m3-short and m3/alpha(2)-short). Although co-transfection of al pha(2)/m3 and m3/alpha(2) resulted in the appearance of specific bindi ng, the co-expression of the two short constructs (alpha(2)/m3-short a nd m3/alpha(2)-short), either together or in combination, respectively , with m8/alpha(2) and alpha(2)/m3 did not result in any detectable bi nding activity, In another set of experiments, a mutant m3 receptor, m 3/m2(16aa), containing 16 amino acids of the m2 receptor sequence at t he amino terminus of the third cytoplasmic loop, which was capable of binding muscarinic ligands but was virtually unable to stimulate phosp hatidylinositol 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 (m8-trunc, containing a n in frame stop codon after amino acid codon 272 of the rat m3 sequenc e) resulted in a considerable carbachol-stimulated phosphatidylinosito l breakdown, the co-transfection of m3/m2(16aa) short with the truncat ed form of the m3 receptor did not result in any recovery of the funct ional activity, Thus, these data suggest that intermolecular interacti on between muscarinic receptors, involving the exchange of amino-termi nal (containing TM domains I-V) and carboxyl-terminal (containing TRI domains VI and MI) receptor fragments depends on the presence of a lon g ia loop, One may speculate that when alternative forms of receptors with a different length of the i3 loop exist, they could have a differ ent propensity to dimerize.