Activation of constitutive 5-hydroxytryptamine1B receptor by a series of mutations in the BBXXB motif: positioning of the third intracellular loop distal junction and its Goα protein interactions

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Abstract
Constitutive activity of the recombinant human 5-hydroxytryptamine1B (5-HT1B) receptor (RC code 2.1.5HT.01.B) was investigated by mutagenesis of the BBXXB motif (in which B represents a basic residue and X a non-basic residue) located in the C-terminal portion of the third intracellular loop. In contrast with wild-type 5-HT1B receptors, three receptor mutants (Thr313 → Lys, Thr313 → Arg and Thr313 → Gln) increased their agonist-independent guanosine 5′-[γ-[35S]thio]triphosphate binding response by 26-41%. This activity represented approx. 30% of the maximal response induced by 5-HT and could be reversed by the inverse agonists methiothepin and 3-(3-dimethylaminopropyl)-4-hydroxy-N-(4-pyridin-4-yl phenyl)-benzenamide (GR 55562). Enhanced agonist-independent and agonist-dependent 5-HT1B receptor activation was provided by co-expression of a pertussis toxin-resistant rat Goα Cys351 → Ile protein. The wild-type 5-HT1B receptor displayed a doubling in basal activity, whereas a spectrum of enhanced basal activities (313-571%) was observed with a series of diverse amino acid substitutions (isoleucine, glycine, asparagine, alanine, lysine, phenylalanine, glutamine and arginine) at the 5-HT1B receptor position 313 in the presence of pertussis toxin (100 ng/ml). Consequently, the constitutive 5-HT1B receptor activity can be modulated by the mutation of Thr313, and displays a graded range between 11% and 59% of maximal 5-HT1B receptor activation by 5-HT. No clear pattern is apparent in the framework of traditionally cited amino acid characteristics (i.e. residue size, charge or hydrophobicity) to explain the observed constitutive activities. The various amino acid substitutions that yielded enhanced activity are unlikely to make similar intramolecular interactions within the 5-HT1B receptor. It is hypothesized that the positioning of the junction between the third intracellular loop and transmembrane domain VI is altered by mutation of Thr313 in the BBXXB motif and thereby unmasks Gα-protein interaction points.

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