Engineering G protein-coupled receptors to facilitate their structure determination

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Over the last two years, 10 new high-resolution structures of G protein-coupled receptors (GPCRs), either with antagonist bound or in an active-like state, have been solved. Whilst the structures of bovine opsin and squid rhodopsin were determined using protein purified from native sources, a rhodopsin mutant structure, the structures of the β1 and β2 adrenergic receptors and the adenosine A2a receptor were determined from engineered protein heterologously expressed in either insect or mammalian cells. These results are the culmination of years of careful work and have resulted in three new strategies for structure determination of GPCRs that can be applied to virtually any membrane protein. Structural and functional investigations have defined a number of conserved interaction networks between key residues in GPCRs that are probably important for receptor structure and activation. Recent evidence indicates that these networks could be disrupted and rearranged independently from each other, suggesting a possible mechanism for full and partial receptor activation. In addition, one of the opsin structures suggests how one of the highest conserved residues in GPCRs, Arg1353.50 of the (E/D)RY motif in TM3, interacts directly with a bound peptide derived from the carboxy terminus of the alpha-subunit of the G protein (Gαt). This result sets the basis for the elucidation of the relationship between the conformational changes in the receptor and activation of the G protein.

Introduction

The human genome encodes more than 800 G protein-coupled receptors (GPCRs) and about 350 are non-odorant receptors with potential medical relevance [1]. These proteins transduce extracellular signals across the cell membrane by activating cytoplasmic heterotrimeric GTP binding proteins (G proteins), which, in turn, modulate the activity of downstream effectors. GPCRs mediate the response to very diverse ligands, including hormones and neurotransmitters, as well as light, smell and taste. Despite the large repertoire of different ligands, the conformational changes induced upon agonist binding that lead to receptor activation are highly conserved [2, 3].

GPCRs often have an extensive and complex pharmacology. Whilst agonists fully activate the receptor, partial agonists only induce submaximal activation even at saturating concentrations. On the other hand, whilst antagonists have no effect on basal ligand-independent activity, inverse agonists are able to decrease it. Therefore, on the basis of functional behaviour, GPCRs behave like rheostats, where different ligands dictate any level of activity [4]. However, there are additional complexities because a single GPCR ligand may affect various signalling pathways in different manners, a concept recently defined as ‘pluridimensional efficacy’ [5]. For example, in the β2 adrenergic receptor (β2AR) some inverse agonists of G protein activation have been shown to be agonists for the beta arrestin ERK1/2 pathway [6]. This is consistent with a growing body of experimental evidence for the existence of multiple conformational states along the activation process of GPCRs [7]. Within this framework, each ligand may induce or stabilise a unique conformational state that can be distinguished by the activity of that state towards different signalling molecules [4], in turn associated with different biochemical cascades and physiological responses. In this way, a ligand can cause the receptor to express some, but not all, of its repertoire of activities towards the cell; this effect is known as collateral efficacy [8], and links ligand binding, receptor structure and biological activity.

Structural studies on GPCRs have, unfortunately, lagged behind the pace at which GPCRs have been functionally characterised, despite significant interest from the pharmaceutical industry where marketed drugs targeting GPCRs earn billions of dollars per annum in world-wide sales [9]. Electron crystallography of two-dimensional rhodopsin crystals first resolved the packing and orientation of the transmembrane helices [10, 11, 12, 13], which provided a template for subsequent GPCR models [2]. In 2000, the first X-ray structure of rhodopsin was determined [14] and, until 2007, rhodopsin was the only GPCR whose structure had been determined to near atomic resolution [14, 15, 16]; the first structure of a recombinant GPCR, rhodopsin thermostabilised with an engineered disulfide bridge, was also published in 2007. These structures allowed the development of more detailed models of the inactive ground state of other GPCRs [17], but gave little insight into the loop regions of other GPCRs and structural changes that occurred upon activation. However, in the last two years there has been a dramatic increase in our understanding of the structure and function of GPCRs, based upon the elucidation of 10 new structures (Table 1). These include the structures of a recombinant thermostabilised rhodopsin [18], three different hormone receptors [19••, 20••, 21, 22••, 23••], the first Gq-coupled receptor [24, 25], and bovine opsin in an active-like state [26•, 27••]. In the rest of this review, we will focus on the new strategies devised to solve the structures of the hormone receptors and the new insights all of these structures have given to GPCR biology.

Section snippets

Improving methodologies for GPCR structure determination

There are many significant challenges that need to be overcome before the structure of a GPCR can be determined, and much attention has been given to problems in the steps of overexpression, purification and crystallisation. However, although many GPCRs were overexpressed over 15 years ago [28], few have been crystallised, despite efforts to remove flexible regions of the protein and ensuring that any post-translational modifications are homogeneous. What is now clear is that the lack of

Structural and functional insights from GPCR structures

The recent GPCR structures have been extensively reviewed [42, 43, 44, 45], but some additional brief comments may prove useful. A comparison of the structures of rhodopsin [14, 15, 16] and the hormone receptors β1AR [22••], β2AR [19••, 20••, 22••, 35••] and A2aR [23••], shows that the helical framework of these GPCRs is well conserved. However, the positions of helical irregularities within the transmembrane domains seem to be more conserved than the relative arrangement of the helices. In

References and recommended reading

Paper of particular interest, published within the period of review, have been highlighted as:

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  • •• of outstanding interest

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