Trends in Neurosciences
Volume 31, Issue 2, February 2008, Pages 74-81
Journal home page for Trends in Neurosciences

Review
GPCR monomers and oligomers: it takes all kinds

https://doi.org/10.1016/j.tins.2007.11.007Get rights and content

Accumulating evidence of G-protein-coupled receptor (GPCR) oligomerization on the one hand and perfect functionality of monomeric receptors on the other creates an impression of controversy. However, the GPCR superfamily is extremely diverse, both structurally and functionally. The life cycle of each receptor includes many stages: synthesis, quality control in the endoplasmic reticulum, maturation in the Golgi, delivery to the plasma membrane (where it can be in the inactive or active state, in complex with cognate G protein, G-protein-coupled receptor kinase or arrestin), endocytosis and subsequent sorting in endosomes. Different GPCR subtypes, and even the same receptor at different stages of its life cycle, most likely exist in different oligomerization states, from monomers to dimers and possibly higher-order oligomers.

Introduction

G-protein-coupled receptors (GPCRs) are the largest family of signaling proteins, encoded in animals by 3%–5% of all genes. Mammals have ∼800–1000 GPCR subtypes, some of which have several forms generated by alternative splicing or mRNA editing. GPCRs have a structurally homologous core of seven transmembrane α helices, but the size and structure of their extracellular and intracellular elements vary wildly, from the most ‘compact’ rhodopsin to receptors with huge extracellular N termini, and others with very large third intracellular loops or C termini [1].

Despite this structural diversity, the first round of signaling initiated by most GPCRs is remarkably uniform: the active receptor catalyzes the exchange of GDP for GTP on heterotrimeric G proteins, whereupon Gα-GTP and released Gβγ activate or inhibit various effectors [1]. Active GPCRs are specifically phosphorylated by G-protein-coupled receptor kinases (GRKs) [2]. Preferential binding of arrestins to active phosphoreceptors precludes further G protein activation [3]. Receptor-bound arrestin recruits two components of internalization machinery, clathrin [4] and AP-2 [5], and a surprising variety of other proteins, initiating the second round of signaling 6, 7.

GPCRs respond to various external stimuli (light, odorants) and signals within the body (hormones, neurotransmitters, extracellular Ca2+, protease activity) and are targeted by half of clinically used drugs. The unrivaled biological significance of GPCRs explains the enormous efforts that have been invested into the elucidation of the mechanisms of their function. Although there are many outstanding issues in this area, one question recently came to the fore and became a subject of fierce debate 8, 9: do GPCRs function as monomers, dimers or higher-order oligomers? Here we show that this is, in fact, a series of questions that do not necessarily have the same answer for all GPCRs, or even for a single receptor at different stages of its life cycle.

Section snippets

The birth of the receptor

Like all integral membrane proteins, GPCRs are synthesized in the rough endoplasmic reticulum. Nascent receptors have to pass numerous steps of quality control and posttranslational modifications before they are delivered to the plasma membrane and get their first chance to function. This process has been extensively studied for class C GPCRs. These receptors have two unique properties: they are known to be obligatory dimers 10, 11, 12, and their ligand-binding site is localized on the

The life in the plasma membrane

GPCRs might be delivered to their ‘place of employment’ as either monomers or oligomers; we only have an unambiguous answer for class C receptors. Regardless of the form in which they arrive, there remain five questions about their self-association status in the plasma membrane, the answers to which might be different for different receptors and even for a single receptor subtype in different functional states. We need to establish in what form the receptor exists when it is: (i) inactive; (ii)

What is the G-protein-activating unit?

It is important to note that the state of inactive receptor does not provide the answer to this question: upon activation, dimers might dissociate 30, 31 or monomers associate 32, 33. Unfortunately, there are no experimental tools to address the most relevant issue: the state of endogenous receptors activating their cognate G proteins in living cells (Figure 1). Therefore, experimentally tractable related questions are asked: first, is a single GPCR molecule sufficient for G protein activation,

Receptor phosphorylation

The mechanism of GRK action provides a perfect opportunity to test whether receptor kinases preferentially phosphorylate GPCR monomers or oligomers. The enzymatic activity of GRK toward exogenous substrates is greatly increased upon receptor binding, indicating that the active receptor serves as both the GRK activator and as its substrate [41]. In rod disc membranes, where rhodopsin molecules cover ∼50% of the surface, light activation of a few rhodopsins results in phosphorylation of a large

Arrestin binding: stoichiometry matters

One of the arguments advanced in support of GPCR oligomerization was that the fit between known structures of rhodopsin 44, 45 and arrestin 46, 47, 48 could be best achieved if one arrestin bound two rhodopsins in a dimer [29]. In bright light, arrestin moves to the rhodopsin-containing outer segments of photoreceptor cells and remains there as long as binding-competent rhodopsin is present [49]. Thus, the extent of arrestin translocation provides a quantitative measure of arrestin–rhodopsin

Arrestin-mediated signaling: could receptor oligomers help?

Receptor-bound arrestins serve as scaffolds mobilizing an astonishing variety of signaling proteins 7, 58. The relative size of arrestins and their binding partners suggests that a unitary arrestin–receptor complex can accommodate no more than four to six additional proteins simultaneously [59]. Thus, either there is stiff competition among arrestin-binding proteins for very limited ‘parking space’ (Figure 3a), or the scaffolds include more than one arrestin–receptor complex. Conceivably,

In what form are GPCRs internalized?

With the exception of class C receptors, we do not have incontrovertible evidence regarding the oligomerization state of any GPCR at this stage of its life cycle. Several studies suggest that some receptors internalize as oligomers 22, 64, 65, but these results could also be rationalized without invoking receptor oligomerization. Several types of GPCRs reside in microdomains covering a small fraction of the cell surface 16, 66, 67. Crowding of overexpressed receptors in these microdomains is

Do all GPCRs function the same way?

An implicit assumption in the current debate between ‘monomer’ and ‘dimer’ parties 8, 9 is that the functional state of all GPCRs remains the same throughout their entire life cycle. However, there is no reason to believe that even a single receptor passes quality control in the endoplasmic reticulum, is delivered to the plasma membrane, exists there before and after activation, binds G protein, GRK, arrestin, is internalized and then undergoes sorting in endosomes in the same oligomerization

How can we get unambiguous answers?

Another problem that plagues the monomer–dimer debate is the unambiguous interpretation of inherently ambiguous data. Functional crosstalk, energy transfer and receptor crosslinking all allow alternative interpretations. There are very few experimental approaches that can yield clear answers in living cells. The only reason we are fairly sure how class C GPCRs work is that in this case only strictly defined heterodimers can make it to the plasma membrane and function 10, 11, 12, which is

Acknowledgements

This work was supported by NIH grants EY011500, GM077561 (V.V.G.) and NS045117 (E.V.G.).

Glossary

Arrestins
A family of four proteins in mammals that specifically bind active phosphorylated GPCRs, shut down (‘arrest’) G-protein-mediated signaling, promote receptor internalization by linking it to the clathrin coat and redirect the signaling to multiple G-protein-independent pathways.
G-protein-coupled receptors (GPCRs)
A large family of receptors (encoded by >800 genes in the human genome) that have in common a characteristic bundle of seven membrane-spanning α helices (heptahelical domain)

References (70)

  • M. Grant

    Agonist-dependent dissociation of human somatostatin receptor 2 dimers: a role in receptor trafficking

    J. Biol. Chem.

    (2004)
  • S. Cvejic et al.

    Dimerization of the δ opioid receptor: implication for a role in receptor internalization

    J. Biol. Chem.

    (1997)
  • T.E. Hebert

    A peptide derived from a β2-adrenergic receptor transmembrane domain inhibits both receptor dimerization and activation

    J. Biol. Chem.

    (1996)
  • B. Jastrzebska

    Functional and structural characterization of rhodopsin oligomers

    J. Biol. Chem.

    (2006)
  • T.H. Bayburt

    Transducin activation by nanoscale lipid bilayers containing one and two rhodopsins

    J. Biol. Chem.

    (2007)
  • J.L. Baneres et al.

    Structure-based analysis of GPCR function: evidence for a novel pentameric assembly between the dimeric leukotriene B4 receptor BLT1 and the G-protein

    J. Mol. Biol.

    (2003)
  • K. Palczewski

    Mechanism of rhodopsin kinase activation

    J. Biol. Chem.

    (1991)
  • B.M. Binder

    Light activation of one rhodopsin molecule causes the phosphorylation of hundreds of others. A reaction observed in electropermeabilized frog rod outer segments exposed to dim illumination

    J. Biol. Chem.

    (1990)
  • G.W. Shi

    Light causes phosphorylation of nonactivated visual pigments in intact mouse rod photoreceptor cells

    J. Biol. Chem.

    (2005)
  • J. Li

    Structure of bovine rhodopsin in a trigonal crystal form

    J. Mol. Biol.

    (2004)
  • J.A. Hirsch

    The 2.8 Å crystal structure of visual arrestin: a model for arrestin's regulation

    Cell

    (1999)
  • M. Han

    Crystal structure of β-arrestin at 1.9 Å: possible mechanism of receptor binding and membrane translocation

    Structure

    (2001)
  • R.B. Sutton

    Crystal structure of cone arrestin at 2.3 Å: evolution of receptor specificity

    J. Mol. Biol.

    (2005)
  • K.S. Nair

    Light-dependent redistribution of arrestin in vertebrate rods is an energy-independent process governed by protein-protein interactions

    Neuron

    (2005)
  • J. Celver

    Conservation of the phosphate-sensitive elements in the arrestin family of proteins

    J. Biol. Chem.

    (2002)
  • A. Kovoor

    Targeted construction of phosphorylation-independent β-arrestin mutants with constitutive activity in cells

    J. Biol. Chem.

    (1999)
  • S.A. Vishnivetskiy

    Mapping the arrestin-receptor interface: structural elements responsible for receptor specificity of arrestin proteins

    J. Biol. Chem.

    (2004)
  • S.A. Vishnivetskiy

    How does arrestin respond to the phosphorylated state of rhodopsin?

    J. Biol. Chem.

    (1999)
  • V.V. Gurevich

    Arrestin interaction with G protein-coupled receptors. Direct binding studies of wild type and mutant arrestins with rhodopsin, β2-adrenergic, and m2 muscarinic cholinergic receptors

    J. Biol. Chem.

    (1995)
  • V.V. Gurevich et al.

    The structural basis of arrestin-mediated regulation of G protein-coupled receptors

    Pharmacol. Ther.

    (2006)
  • Y.M. Kim et al.

    Differential roles of arrestin-2 interaction with clathrin and adaptor protein 2 in G protein-coupled receptor trafficking

    J. Biol. Chem.

    (2002)
  • Y. Daaka

    Essential role for G protein-coupled receptor endocytosis in the activation of mitogen-activated protein kinase

    J. Biol. Chem.

    (1998)
  • J.L. DeGraff

    Role of arrestins in endocytosis and signaling of α2-adrenergic receptor subtypes

    J. Biol. Chem.

    (1999)
  • P.C. Brum

    Differential targeting and function of α2A and α2C adrenergic receptor subtypes in cultured sympathetic neurons

    Neuropharmacology

    (2006)
  • J.P. Vilardaga

    Differential conformational requirements for activation of G proteins and the regulatory proteins arrestin and G protein-coupled receptor kinase in the G protein-coupled receptor for parathyroid hormone (PTH)/PTH-related protein

    J. Biol. Chem.

    (2001)
  • Cited by (0)

    View full text