An inhibitory compound against the interaction between G alpha(s) and the third intracellular loop region of serotonin receptor subtype 6 (5-HT6) disrupts the signaling pathway of 5-HT6.

Serotonin receptor subtype 6 (5-HT(6)) is a neurotransmitter receptor, which is involved in various brain functions such as memory and mood. It mediates signaling via the interaction with a stimulatory G-protein. Especially, the third intracellular loop (iL3) of 5-HT(6) and the alpha subunit of stimulatory G protein (G alpha(s)) are responsible for the signaling process of 5-HT(6). Chemical compounds that could inhibit the interaction between the iL3 region of 5-HT(6) and G alpha(s) were screened from a chemical library consisted of 5,600 synthetic compounds. One of the identified compounds bound to G alpha(s) and effectively blocked the interaction between G alpha(s) and the iL3 region of 5-HT(6). The identified compound was further shown to reduce the serotonin-induced accumulation of cAMP in 293T cells transformed with 5-HT(6) cDNA. It also lowered the Ca(2+) efflux induced by serotonin in cells expressing 5-HT(6) and chimeric G alpha(s5/q). These results indicate that the interaction between the iL3 of 5-HT(6) and G alpha(s) can be exploited for screening of regulatory compounds against the signaling pathway of 5-HT(6).

Molecular analysis of the interaction between the intracellular loops of the human serotonin receptor type 6 (5-HT6) and the alpha subunit of GS protein.

The serotonin type 6 (5-HT(6)) receptor is a G-protein coupled receptor (GPCR) coupled to a stimulatory G-protein (G(S)). To identify the structural basis for the interaction of the 5-HT(6) receptor with the G(S) protein, we have dissected the interaction between GST-fusion proteins containing the second intracellular loop (iL2), the third intracellular loop (iL3), or the C-terminal tail of the 5-HT(6) receptor and the alpha subunit of G(S) (Galpha(S)). The direct interaction of iL3 and Galpha(S) was demonstrated by co-immunoprecipitation. Furthermore, the kinetic parameters of the interaction between iL3 and Galpha(S) were measured by surface plasmon resonance, and the apparent dissociation constant was determined to be 0.9 x 10(-6)M. In contrast, the second intracellular loop and C-terminal tail regions showed negligible affinity to Galpha(S). The critical residues within the iL3 region for the interaction with Galpha(S) were identified as conserved positively charged residues near the C-terminus of iL3 by measuring the cellular levels of cAMP produced in response to 5-HT stimulation of cells transfected with 5-HT(6) receptor mutants.

Identification and characterization of a novel angiostatin-binding protein by the display cloning method.

Angiostatin is a potent anti-angiogenic protein. To examine the angiostatin-interacting proteins, we used the display-cloning method with a T7 phage library presenting human cDNAs. The specific T7 phage clone that bound to the immobilized angiostatin was isolated, and a novel gene encoding the displayed polypeptide on the isolated T7 phage was identified. The displayed angiostatin-binding sequence was expressed in E. coli as a soluble protein and purified to homogeneity. This novel angiostatin-binding region interacted specifically to angiostatin with a dissociation constant of 3.4 x 10(-7) M. A sequence analysis showed that the identified sequence was a part of the large ORF of 1,998 amino acids, whose function has not yet been characterized. A Northern analysis indicated that the gene containing the angiostatin-binding sequence was expressed differentially in the developmental stages or cell types.