Abstract
5-HT2A serotonin receptors are unusual among G-protein coupled receptors in that they can be internalized and desensitized, in some cell types, in an arrestin-independent manner. The molecular basis of the arrestin-insensitivity of 5-HT2A receptors is unknown but is probably caused, in part, by the apparent lack of agonist-induced 5-HT2A receptor phosphorylation. Because the arrestin-insensitivity of 5-HT2A receptors is cell-type selective, we used a “constitutively active” arrestin mutant that can interact with agonist-activated but nonphosphorylated receptors. We show here that this “constitutively active” arrestin mutant (Arr2-R169E) can force 5-HT2A receptors to be regulated by arrestins. Cotransfection of 5-HT2A receptors with Arr2-R169E induced agonist-independent 5-HT2A receptor internalization, and a constitutive translocation of the Arr2-R169E mutant to the plasma membrane, whereas wild-type Arrestin-2 had no effect. Additionally, Arr2-R169E, unlike wild-type arrestin-2, induced a significant decrease in efficacy of agonist-induced phosphoinositide hydrolysis with an unexpected increase in agonist potency. Radioligand binding assays demonstrated that the fraction of receptors in the high-affinity agonist binding-state increased with expression of Arr2-R169E, indicating that Arr2-R169E stabilizes the agonist-high affinity state of the 5-HT2A receptor (R*). Intriguingly, the agonist-independent interaction of Arr2-R169E with 5-HT2A receptors was inhibited by inverse agonist treatment and is thus probably caused by the high level of 5-HT2Areceptor constitutive activity. This is the first demonstration that a constitutively active arrestin mutant can both induce agonist-independent internalization and stabilize the agonist-high affinity state of an arrestin-insensitive G protein coupled receptor.
Footnotes
-
This work was supported in part by National Institutes of Health grants R01-MH61887, R01-MH57635, and K02-MH01366 (to B.L.R.) and R01-GM63097 and R01-EY11500 (to V.V.G.). J.A.G. was supported in part by the National Institutes of Health Medical Scientist Training Program Grant 5T32-GM07250 and Metabolism Training Program grant 5T32-DK07319.
- Abbreviations:
- 5-HT
- 5-hydroxytryptamine
- GPCR
- G protein-coupled receptor
- HEK
- human embryonic kidney
- DOI
- 2,5-dimethoxy-4-iodophenylisopropylamine
- BODIPY-FL
- 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoic acid, succinimidyl ester
- DMEM
- Dulbecco's modified Eagle's medium
- PBS
- phosphate-buffered saline
- PI
- phosphoinositide
- DynK44A
- Dynamin K44A
- GRK
- G protein-coupled receptor kinase
- Arr2-wt
- Arrestin-2
- Arr2-R169E
- Arrestin-2 (R169E)
- Received September 23, 2002.
- Accepted January 22, 2003.
- The American Society for Pharmacology and Experimental Therapeutics
MolPharm articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|