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

被引:8
|
作者
Thiagaraj, Harish V.
Ortiz, Thomas C.
Devereaux, Marvin C., Jr.
Seaver, Ben
Hall, Brian
Parker, Keith K.
机构
[1] Univ Montana, Skaggs Sch Pharm, Dept Biomed & Pharmaceut Sci, Missoula, MT 59812 USA
[2] Univ Montana, Ctr Struct & Funct Neurosci, Missoula, MT 59812 USA
关键词
serotonin; 5HT1a receptor; G proteins; synthetic peptides; cyclic AMP; 35S]-gamma-S-GTP;
D O I
10.1016/j.neuint.2006.07.017
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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, PI I was active in all three receptor preparations tested: whole cells, membrane bound, and solubilized. In analyses of the membrane bound receptor system, PI I 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, PI I 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. (c) 2006 Elsevier Ltd. All rights reserved.
引用
收藏
页码:109 / 118
页数:10
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