Regulation of G proteins by human 5-HT1a receptor TM3/i2 and TM5/i3 loop peptides.
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]gamma-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]gamma-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.
Thiagaraj
Harish V
HV
Department of Biomedical and Pharmaceutical Sciences (MPH I02), Skaggs School of Pharmacy, The University of Montana, 32 Campus Drive #1552, Missoula, MT 59812-1552, United States.
Ortiz
Thomas C
TC
Devereaux
Marvin C
MC
Seaver
Ben
B
Hall
Brian
B
Parker
Keith K
KK
D000595:Amino Acid Sequence; D000242:Cyclic AMP / Q000737:chemistry / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism*; D044282:Receptor, Serotonin, 5-HT1A / Q000737:chemistry / Q000378:metabolism*
D010446:Peptide Fragments; D044282:Receptor, Serotonin, 5-HT1A; D000242:Cyclic AMP; D019204:GTP-Binding Proteins