Regulation of G proteins by human 5-HT1a receptor TM3/i2 and TM5/i3 loop peptides

• Published in: Neurochemistry international 2007, Vol.50 (1), p.109-118
• Main Authors: Thiagaraj, Harish V., Ortiz, Thomas C., Devereaux, Marvin C., Seaver, Ben, Hall, Brian, Parker, Keith K.
• Format: Article
• Language: English
• Subjects: 5HT1a receptor; [35S]γ-S-GTP; Amino Acid Sequence; Cyclic AMP; Cyclic AMP - chemistry; Cyclic AMP - metabolism; G proteins; GTP-Binding Proteins - metabolism; Humans; Molecular Sequence Data; Peptide Fragments - metabolism; Receptor, Serotonin, 5-HT1A - chemistry; Receptor, Serotonin, 5-HT1A - metabolism; Serotonin; Synthetic peptides
• ISSN: 0197-0186; 1872-9754
• DOI: 10.1016/j.neuint.2006.07.017

A bioactive synthetic 11 amino acid peptide probe (P11) was constructed according to the published sequence of the human 5HT1a receptor. The probe was used to enhance understanding of cytoplasmic loop 2/G protein coupling and activation. Additionally, two peptides (P8, P9) from the cytoplasmic loop 3 region were synthesized and studied. These probes were tested in a model system of human 5HT1a receptor stably expressed in Chinese Hamster Ovary cells. In agonist inhibition studies, P11 was active in all three receptor preparations tested: whole cells, membrane bound, and solubilized. In analyses of the membrane bound receptor system, P11 demonstrated uncompetitive inhibition characteristics. When forskolin-stimulated cAMP levels were measured, P11 was inactive in this negatively coupled system. Utilizing a [35S]γ-S-GTP incorporation assay, P11 was unable to stimulate G protein incorporation of GTP. While P8 and P9 were also broadly active as non-competitive agonist inhibitors, their characteristics differed in the signal transduction system. P8 and P9 did not significantly change forskolin-stimulated cAMP levels. However, P8 increased [35S]γ-S-GTP incorporation, while P9 decreased incorporation. Thus, P11, a synthetic peptide from the TM3/i2 region of the receptor, provides suggestive evidence that this receptor region is involved in G protein coupling but not activation. On the other hand, P8 and P9 activities suggest that the TM5/i3 region is involved in both coupling to and regulation of G protein activity. The current evidence from these cytoplasmic loop regions is discussed in the overall context of an emerging model for human 5HT1a receptor–G protein interactions.