Structure and function of the third intracellular loop of the 5- hydroxytryptamine(2A) receptor: The third intracellular loop is α-helical and binds purified arrestins

Edward I. Gelber, Wesley K. Kroeze, David L. Willins, John Gray, Christine A. Sinar, Edward G. Hyde, Vsevolod Gurevich, Jeffrey Benovic, Bryan L. Roth

Research output: Contribution to journalArticle

57 Citations (Scopus)

Abstract

Understanding the precise structure and function of the intracellular domains of G protein-coupled receptors is essential for understanding how receptors are regulated, and how they transduce their signals from the extracellular milieu to intracellular sites. To understand better the structure and function of the intracellular domain of the 5- hydroxytryptamine(2A) (5-HT(2A)) receptor, a model G(∞&q)-coupled receptor, we overexpressed and purified to homogeneity the entire third intracellular loop (i3) of the 5-HT(2A) receptor, a region previously implicated in G- protein coupling. Circular dichroism spectroscopy of the purified i3 protein was consistent with α-helical and β-loop, -turn, and -sheet structure. Using random peptide phage libraries, we identified several arrestin-like sequences as i3-interacting peptides. We subsequently found that all three known arrestins (β-arrestin, arrestin-3, and visual arrestin) bound specifically to fusion proteins encoding the i3 loop of the 5-HT(2A) receptor. Competition binding studies with synthetic and recombinant peptides showed that the middle portion of the i3 loop, and not the extreme N and C termini, was likely to be involved in i3-arrestin interactions. Dual-label immunofluorescence confocal microscopic studies of rat cortex indicated that many cortical pyramidal neurons coexpressed arrestins (β-arrestin or arrestin-3) and 5-HT(2A) receptors, particularly in intracellular vesicles. Our results demonstrate (a) that the i3 loop of the 5-HT(2A) receptor represents a structurally ordered domain composed of α-helical and β-loop, -turn, and -sheet regions, (b) that this loop interacts with arrestins in vitro, and is hence active, and (c) that arrestins are colocalized with 5- HT(2A) receptors in vivo.

Original languageEnglish (US)
Pages (from-to)2206-2214
Number of pages9
JournalJournal of Neurochemistry
Volume72
Issue number5
DOIs
StatePublished - 1999
Externally publishedYes

Fingerprint

Arrestins
Arrestin
Serotonin Receptors
Serotonin
Peptides
Receptor, Serotonin, 5-HT2A
Peptide Library
Pyramidal Cells
Circular dichroism spectroscopy
G-Protein-Coupled Receptors
Circular Dichroism
Bacteriophages
GTP-Binding Proteins
Fluorescent Antibody Technique
Spectrum Analysis
Proteins
Neurons
Rats
Labels
Fusion reactions

Keywords

  • β-Arrestin
  • 5-HT(2A) receptor
  • Receptor-effector coupling
  • Structure-function

ASJC Scopus subject areas

  • Biochemistry
  • Cellular and Molecular Neuroscience

Cite this

Structure and function of the third intracellular loop of the 5- hydroxytryptamine(2A) receptor : The third intracellular loop is α-helical and binds purified arrestins. / Gelber, Edward I.; Kroeze, Wesley K.; Willins, David L.; Gray, John; Sinar, Christine A.; Hyde, Edward G.; Gurevich, Vsevolod; Benovic, Jeffrey; Roth, Bryan L.

In: Journal of Neurochemistry, Vol. 72, No. 5, 1999, p. 2206-2214.

Research output: Contribution to journalArticle

Gelber, Edward I. ; Kroeze, Wesley K. ; Willins, David L. ; Gray, John ; Sinar, Christine A. ; Hyde, Edward G. ; Gurevich, Vsevolod ; Benovic, Jeffrey ; Roth, Bryan L. / Structure and function of the third intracellular loop of the 5- hydroxytryptamine(2A) receptor : The third intracellular loop is α-helical and binds purified arrestins. In: Journal of Neurochemistry. 1999 ; Vol. 72, No. 5. pp. 2206-2214.
@article{cf7c3bd5057c45709b0f8e58db1384c4,
title = "Structure and function of the third intracellular loop of the 5- hydroxytryptamine(2A) receptor: The third intracellular loop is α-helical and binds purified arrestins",
abstract = "Understanding the precise structure and function of the intracellular domains of G protein-coupled receptors is essential for understanding how receptors are regulated, and how they transduce their signals from the extracellular milieu to intracellular sites. To understand better the structure and function of the intracellular domain of the 5- hydroxytryptamine(2A) (5-HT(2A)) receptor, a model G(∞&q)-coupled receptor, we overexpressed and purified to homogeneity the entire third intracellular loop (i3) of the 5-HT(2A) receptor, a region previously implicated in G- protein coupling. Circular dichroism spectroscopy of the purified i3 protein was consistent with α-helical and β-loop, -turn, and -sheet structure. Using random peptide phage libraries, we identified several arrestin-like sequences as i3-interacting peptides. We subsequently found that all three known arrestins (β-arrestin, arrestin-3, and visual arrestin) bound specifically to fusion proteins encoding the i3 loop of the 5-HT(2A) receptor. Competition binding studies with synthetic and recombinant peptides showed that the middle portion of the i3 loop, and not the extreme N and C termini, was likely to be involved in i3-arrestin interactions. Dual-label immunofluorescence confocal microscopic studies of rat cortex indicated that many cortical pyramidal neurons coexpressed arrestins (β-arrestin or arrestin-3) and 5-HT(2A) receptors, particularly in intracellular vesicles. Our results demonstrate (a) that the i3 loop of the 5-HT(2A) receptor represents a structurally ordered domain composed of α-helical and β-loop, -turn, and -sheet regions, (b) that this loop interacts with arrestins in vitro, and is hence active, and (c) that arrestins are colocalized with 5- HT(2A) receptors in vivo.",
keywords = "β-Arrestin, 5-HT(2A) receptor, Receptor-effector coupling, Structure-function",
author = "Gelber, {Edward I.} and Kroeze, {Wesley K.} and Willins, {David L.} and John Gray and Sinar, {Christine A.} and Hyde, {Edward G.} and Vsevolod Gurevich and Jeffrey Benovic and Roth, {Bryan L.}",
year = "1999",
doi = "10.1046/j.1471-4159.1999.0722206.x",
language = "English (US)",
volume = "72",
pages = "2206--2214",
journal = "Journal of Neurochemistry",
issn = "0022-3042",
publisher = "Wiley-Blackwell",
number = "5",

}

TY - JOUR

T1 - Structure and function of the third intracellular loop of the 5- hydroxytryptamine(2A) receptor

T2 - The third intracellular loop is α-helical and binds purified arrestins

AU - Gelber, Edward I.

AU - Kroeze, Wesley K.

AU - Willins, David L.

AU - Gray, John

AU - Sinar, Christine A.

AU - Hyde, Edward G.

AU - Gurevich, Vsevolod

AU - Benovic, Jeffrey

AU - Roth, Bryan L.

PY - 1999

Y1 - 1999

N2 - Understanding the precise structure and function of the intracellular domains of G protein-coupled receptors is essential for understanding how receptors are regulated, and how they transduce their signals from the extracellular milieu to intracellular sites. To understand better the structure and function of the intracellular domain of the 5- hydroxytryptamine(2A) (5-HT(2A)) receptor, a model G(∞&q)-coupled receptor, we overexpressed and purified to homogeneity the entire third intracellular loop (i3) of the 5-HT(2A) receptor, a region previously implicated in G- protein coupling. Circular dichroism spectroscopy of the purified i3 protein was consistent with α-helical and β-loop, -turn, and -sheet structure. Using random peptide phage libraries, we identified several arrestin-like sequences as i3-interacting peptides. We subsequently found that all three known arrestins (β-arrestin, arrestin-3, and visual arrestin) bound specifically to fusion proteins encoding the i3 loop of the 5-HT(2A) receptor. Competition binding studies with synthetic and recombinant peptides showed that the middle portion of the i3 loop, and not the extreme N and C termini, was likely to be involved in i3-arrestin interactions. Dual-label immunofluorescence confocal microscopic studies of rat cortex indicated that many cortical pyramidal neurons coexpressed arrestins (β-arrestin or arrestin-3) and 5-HT(2A) receptors, particularly in intracellular vesicles. Our results demonstrate (a) that the i3 loop of the 5-HT(2A) receptor represents a structurally ordered domain composed of α-helical and β-loop, -turn, and -sheet regions, (b) that this loop interacts with arrestins in vitro, and is hence active, and (c) that arrestins are colocalized with 5- HT(2A) receptors in vivo.

AB - Understanding the precise structure and function of the intracellular domains of G protein-coupled receptors is essential for understanding how receptors are regulated, and how they transduce their signals from the extracellular milieu to intracellular sites. To understand better the structure and function of the intracellular domain of the 5- hydroxytryptamine(2A) (5-HT(2A)) receptor, a model G(∞&q)-coupled receptor, we overexpressed and purified to homogeneity the entire third intracellular loop (i3) of the 5-HT(2A) receptor, a region previously implicated in G- protein coupling. Circular dichroism spectroscopy of the purified i3 protein was consistent with α-helical and β-loop, -turn, and -sheet structure. Using random peptide phage libraries, we identified several arrestin-like sequences as i3-interacting peptides. We subsequently found that all three known arrestins (β-arrestin, arrestin-3, and visual arrestin) bound specifically to fusion proteins encoding the i3 loop of the 5-HT(2A) receptor. Competition binding studies with synthetic and recombinant peptides showed that the middle portion of the i3 loop, and not the extreme N and C termini, was likely to be involved in i3-arrestin interactions. Dual-label immunofluorescence confocal microscopic studies of rat cortex indicated that many cortical pyramidal neurons coexpressed arrestins (β-arrestin or arrestin-3) and 5-HT(2A) receptors, particularly in intracellular vesicles. Our results demonstrate (a) that the i3 loop of the 5-HT(2A) receptor represents a structurally ordered domain composed of α-helical and β-loop, -turn, and -sheet regions, (b) that this loop interacts with arrestins in vitro, and is hence active, and (c) that arrestins are colocalized with 5- HT(2A) receptors in vivo.

KW - β-Arrestin

KW - 5-HT(2A) receptor

KW - Receptor-effector coupling

KW - Structure-function

UR - http://www.scopus.com/inward/record.url?scp=0032971464&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0032971464&partnerID=8YFLogxK

U2 - 10.1046/j.1471-4159.1999.0722206.x

DO - 10.1046/j.1471-4159.1999.0722206.x

M3 - Article

C2 - 10217304

AN - SCOPUS:0032971464

VL - 72

SP - 2206

EP - 2214

JO - Journal of Neurochemistry

JF - Journal of Neurochemistry

SN - 0022-3042

IS - 5

ER -