Activation of constitutive 5-hydroxytryptamine(1B) receptor by a series ofmutations in the BBXXB motif: positioning of the third intracellular loop distal junction and its G(0)alpha protein interactions

Constitutive, activity of the recombinant human 5-hydroxy-wtryptamine(1B) ( 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 re sidue) located in the C-terminal portion of the third intracellular loop. I n contrast with:wild-type 5-HT1B re0ceptors, three receptor mutants (Thr(31 3) --> Lys, Thp(313), Arg and Thr(313) --> Gln) increased their agonist-ind ependent guanosine 5'-[gamma-[S-35]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 G(0)alpha Cys(351) --> Ile protein. The wild-type 5-HT1B receptor displaye d a doubling in basal activity, whereas a spectrum of enhanced basal activi ties (313-571%) was observed with a series of diverse amino acid substituti ons (isoleucine, glycine, asparagine, alanine, lysine, phenylalanine, gluta mine and arginine) at the 5-HT1B receptor position 313 in the presence of p ertussis toxin (100 ng/ml). Consequently, the constitutive 5HT(1B) receptor activity can be modulated by the mutation of Thr(313), and displays a grad ed 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 expl ain the observed constitutive activities. The various amino acid substituti ons that yielded enhanced activity are unlikely to make similar intramolecu lar interactions within the 5-HT1B receptor. It is hypothesized that the po sitioning of the junction between the third intracellular loop and transmem brane domain VI is altered by mutation of Thr(313) in the BBXXB motif and t hereby unmasks G(alpha)-protein interaction points.