The aim of this study was to investigate the molecular changes associated with the transition of the human oxytocin receptor from its inactive to its active states. Mutation of the conserved arginine of the glutamate/aspartate-arginine-tyrosine motif located in the second intracellular domain gave rise to the first known constitutively active oxytocin receptor (R137A), whereas mutation of the aspartic acid located in the second transmembrane domain led to an inactive receptor (D85A). The structural features of the constitutively active and inactive receptor mutants were compared with those of the wild type in its free and agonist-bound states. The results suggest that, although differently triggered, the activation process induced by the agonist and the activating mutation are characterized by the opening of a solvent exposed site formed by the 2nd intracellular loop, the cytosolic extension of helix 5, and the 3rd intracellular loop; on the contrary, the D85A mutation prevents oxytocin from triggering the opening of a cytosolic site. On the basis of these findings, we hypothesize that this cytosolic crevice plays an important role in G protein recognition. Finally, comparative analysis of the free- and agonist-bound forms of the wild-type oxytocin receptor and alpha1B adrenergic receptor suggests that the highly conserved polar amino acids and the seven helices play similar mechanistic roles in the different G protein-coupled receptors.

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 ␣ 2 adrenergic/m3 muscarinic receptors in which 196 amino acids were deleted from the i3 loop (␣ 2 /m3-short and m3/␣ 2 -short). Although co-transfection of ␣ 2 /m3 and m3/␣ 2 resulted in the appearance of specific binding, the co-expression of the two short constructs (␣ 2 /m3-short and m3/␣ 2 -short), either together or in combination, respectively, with m3/␣ 2 and ␣ 2 /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.Transmembrane receptors recognize and integrate external signals modifying the metabolism or the ionic equilibrium of the cell milieu. Muscarinic receptors belong to the G-proteincoupled class of receptors (2). Molecular cloning studies have revealed the existence of five structurally related muscarinic receptor proteins (m1-m5; Refs. 3 and 4). The five muscarinic receptors are predicted to be composed of seven hydrophobic transmembrane domains (TM domains I-VII) 1 connected by alternating cytoplasmic and extracellular loops, an extracellular amino-terminal domain and an intracellular carboxyl-terminal segment. These receptors couple to a varied group of effectors, including membrane-associated phospholipases, adenylate and guanylate cyclases, and ion channels (5-8). The third intracellular (i3) loop of these receptors confers specificity for G-protein coupling (9). Moreover, it has been suggested that this segment of the receptor is involved in the phenomenon of internalization and down-regulation (10 -12).In a previous article, Maggio et al. (13) showed that muscarinic receptors behave structurally in a fashion analog...

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