Abstract

The extended classic ternary complex model predicts that a G protein-coupled receptor (GPCR) exists in only two interconvertible states: an inactive R, and an active R^*. However, different structural active R^* complexes may exist in addition to a silent inactive R ground state (Rg). Here we demonstrate, in a cellular context, that several R^* states of 5-hydroxytryptamine-4 (5-HT₄) receptors involve different side-chain conformational toggle switches. Using site-directed mutagenesis and molecular modeling approaches, we show that the basal constitutive receptor (R^*basal) results from stabilization of an obligatory double toggle switch (Thr3.36 from inactive g- to active g+ and Trp6.48 from inactive g+ to active t). Mutation of either threonine or tryptophan to alanine resulted in a lowering of the activity of the R^*basal similar to the Rg. The T3.36A mutation shows that the Thr3.36 toggle switch plays a minor role in the stabilization of R^* induced by 5-HT (R^*-5-HT) and BIMU8 (R^*-BIMU8) and is fully required in the stabilization of R^* induced by (S)-zacopride, cisapride, and 1-(4-amino-5-chloro-2-methoxyphenyl)-3-(1-butyl-4-piperidinyl)-1-propanone (RS 67333) (R^*-benzamides). Thus, benzamides stabilize R^*-benzamides by forming a specific hydrogen bond with Thr3.36 in the active g+ conformation. Conversely, R^*-BIMU8 was probably the result of a direct conformational transition of Trp6.48 from inactive g+ to active t by hydrogen bonding of this residue to a carboxyl group of BIMU8. We were surprised that the Trp6.48 toggle switch was not necessary for receptor activation by the natural agonist 5-HT. R^*-5-HT is probably attained through other routes of activation. Thus, different conformational arrangements occur during stabilization of R^*basal, R^*-5-HT, R^*-benzamides, and R^*-BIMU8.