Trends in Neurosciences
ReviewGPCR monomers and oligomers: it takes all kinds
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)
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