Review
Allosteric sodium in class A GPCR signaling

https://doi.org/10.1016/j.tibs.2014.03.002Get rights and content

Highlights

  • A sodium ion binding site is found in high-resolution structures of class A GPCRs.

  • This allosteric site in the middle of the 7TM domain is highly conserved in class A GPCRs.

  • Specific binding of Na+ explains its biochemical and functional effects on GPCRs.

  • Collapse of the pocket and Na+ relocation are involved in class A GPCR signaling.

Despite their functional and structural diversity, G-protein-coupled receptors (GPCRs) share a common mechanism of signal transduction via conformational changes in the seven-transmembrane (7TM) helical domain. New major insights into this mechanism come from the recent crystallographic discoveries of a partially hydrated sodium ion that is specifically bound in the middle of the 7TM bundle of multiple class A GPCRs. This review discusses the remarkable structural conservation and distinct features of the Na+ pocket in this most populous GPCR class, as well as the conformational collapse of the pocket upon receptor activation. New insights help to explain allosteric effects of sodium on GPCR agonist binding and activation, and sodium's role as a potential co-factor in class A GPCR function.

Section snippets

Early phenomena attributed to a specific sodium-dependent modulation of GPCR function

G-protein-coupled receptors (GPCRs) are the largest superfamily of membrane proteins in the human genome and have key roles in human physiology and in the action of more than 30% of therapeutic drugs [1]. The binding of a ligand stabilizes conformational changes in the receptor, which trigger the activation of intracellular (IC) effectors such as G proteins and arrestins [2], leading to a cascade of cellular responses. Although all GPCRs share a common seven-transmembrane (7TM) architecture,

Identification of Na+ in the crystal structures of class A GPCRs

The recently solved high-resolution (1.8 Å) structure of the A2A adenosine receptor (A2AAR) [8] was the first to reveal a Na+/water cluster in the middle of the 7TM helical bundle, thereby providing a detailed description of a GPCR allosteric site (Figure 1A,B). The Na+ in the A2AAR is coordinated by two highly conserved residues, D2.50 and S3.39, and three water molecules. These water molecules belong to a nearly continuous water-filled passage connecting the A2AAR extracellular (EC) and IC

Distinct types of sodium pockets in inactive GPCRs

The common binding of the Na+ in 7TM bundles, however, does not require an identical pocket structure to that found in the A2AAR and β1AR. Interestingly, opioid receptors (in the γ-branch) present a structural variation on the Na+/water coordination motif, whereas conformations of the 15 conserved residues of the pocket remain similar to A2AAR and β1AR. Indeed, the 1.8 Å resolution structure of the δ-OR [15] (PDB identifier: 4N6H) reveals the pivotal importance of another position, 3.35, for

Conservation of Na+ binding across class A GPCR families and branches

Sequence analysis of all class A GPCRs confirms an exceptionally high conservation of the pocket (Figure 2 and Figure S1 in the supplementary material online). In fact, the pocket (as defined in A2AAR) combines 15 of the 34 residue positions that are conserved in the majority of the non-olfactory class A GPCRs (Figure 2C). The pocket encompasses the three previously identified conserved motifs: (i) F6.44 and W6.48 of the FxxCWxP motif in helix VI; (ii) N7.49 and Y7.53 of the NPxxY motif in

Class A GPCRs lacking a putative Na+ pocket have distinctive properties

Our analysis also suggests that those 36 of the class A receptors (∼5%) that lack acidic residues D(E)2.50 (Table S1 in the supplementary material online) may have functional properties that are distinct from other class A GPCRs and, in most cases, do not possess ligand-modulated signal transduction. Thus, of these 36 proteins, 26 are described as ‘pseudogene’, ‘non-signaling’, ‘decoy’, ‘constitutively active orphan’, or ‘putative/probable’ GPCRs, as annotated by the International Union of

Lack of Na+ binding in visual opsins

Rhodopsin (OPSD) and other visual opsins (OPSB, OPSG, and OPSR) represent an interesting exception that deserves a closer look. The last line in Table S1 shows the absence of D2.50 or any other acidic side chain in the allosteric pocket of blue opsin (OPSB). Moreover, all four opsins lack polar side chains in the other two crucial sodium-coordinating positions, 3.39 and 7.45 (Figure S1 in the supplementary material online), which is likely to abolish specific Na+ binding. Analysis of the

Activation involves structural rearrangements in the Na+ pocket

Further evidence for a key functional role of the Na+ cluster in the modulation of conformational transitions comes from an analysis of active state structures of class A GPCRs (Figure 3). Our comparison of inactive- and active-state crystal structures of A2AAR and β2AR 28, 29 reveals that the Na+ and water pocket collapses in size from ∼200 to <70 Å3 due to the activation-related movements of the TM helices. In particular, an inward movement of helix VII at the NPxxY motif and an outward

Functional studies and challenges

Although biochemical evidence for the allosteric effects of sodium on agonist binding exists for a number of diverse GPCRs (Table 1), a detailed understanding of the functional sodium effect on GPCR signaling in living cells is complicated by several factors. Some evidence for sodium's impact on GPCR function was obtained by direct measurements of G protein binding to isolated cell membranes 31, 32, 33 or G protein-mediated signaling in whole cells 34, 35 as a function of Na+ concentration.

Allosteric sodium site residues modulate signaling bias

Apart from G protein mediated signaling, GPCR function involves other independent pathways, including those mediated by β-arrestin [55]. Selective upregulation or downregulation of these pathways by ligands or mutations often leads to so-called biased signaling or functional selectivity, which is of key importance for GPCR biology and pharmacology 56, 57, 58, 59. Several studies suggested that changes in the allosteric sodium pocket can also lead to pronounced signaling bias: for example, in

Possible mechanisms of sodium as a co-factor in GPCR signaling

An essential role for the Na+ and water cluster in GPCR function is supported by: (i) the structural features of Na+ binding in the center of the 7TM bundle in the inactive state receptors; (ii) an exceptionally high conservation of the pocket in ligand activated class A GPCRs; (iii) dramatic activation-related changes in the Na+ pocket; and (iv) strong allosteric effects of the Na+ on constitutive and ligand-dependent GPCR signaling. One key aspect of the Na+ interactions with GPCRs is that,

Practical implications for GPCR studies

The knowledge of sodium's impact on GPCR function may have immediate practical implications for GPCR structural and functional biology, beyond their early use as a screen for ligand agonism activity [9]. Thus, the allosteric effects of sodium can inform the choice of optimal salt conditions for structural studies. For example, antagonist-bound inactive state receptors have been crystallized in high NaCl concentrations 8, 75, whereas crystallization of agonist-bound receptors in active-like

Concluding remarks

The ‘sodium effect’ on GPCR agonist binding has captivated researchers for more than 40 years. Only now, however, have high-resolution crystallographic studies revealed a common structural basis for Na+-specific binding in the center of the 7TM helical bundle, which explains this and other sodium effects on GPCRs. The observed activation-related collapse of the sodium pocket implicates a specific role for the Na+ in the signal transduction mechanism, where the ion translocates towards or into

Acknowledgments

This work was supported by the National Institute of General Medical Sciences (NIGMS) PSI:Biology grants U54 GM094618 (V.K., V.C., and R.C.S.), U19MH82441 (B.L.R.), and R01DA017204 (B.L.R.). We thank K. Kadyshevskaya for assistance with figure preparation, A. Walker for assistance with manuscript preparation, C. Tate, K. Jacobson, A. IJzerman, and M. Audet for helpful discussions, and C. Tate for providing unreleased coordinates of the 4BVN structure.

Glossary

Allosteric modulation
modification of the orthosteric ligand binding and/or receptor signaling by another ligand or ion that binds to a distinct (allosteric) site.
Ballesteros–Weinstein residue numbering
uses the X.YY format to denote the transmembrane helix number (X) and residue position (YY) relative to the most conserved residue in this helix (X.50). The numbering is used to refer to structurally equivalent residue positions in different G-protein-coupled receptors (GPCRs).
Biased signaling (or

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