Fusion of the third intracellular loop of GPCRs to T4L has proven to be a successful strategy for obtaining well-diffracting crystals of these receptors. Structures of such fusions have been determined both in antagonist- and agonist-bound states (Cherezov et al., 2007
; Rosenbaum et al., 2007
; Jaakola et al., 2008
; Chien et al., 2010
; Wu et al., 2010
; Rasmussen et al., 2011a
; Shimamura et al., 2011
; Xu et al., 2011
; Haga et al., 2012
; Hanson et al., 2012
; Kruse et al., 2012
). However, in no case has such a receptor-T4L fusion been reported to be capable of activating a G protein in a ligand-dependent manner. Furthermore, rigidification of receptors induced by the presence of T4 lysozyme has been invoked to explain apparent anomalies in the behavior of the ‘ionic lock’ between the conserved DRY motif in TM3 and a conserved glutamic acid in TM6 of GPCRs (Dror et al., 2009
; Salon et al., 2011
). Although the ionic interactions comprising the ‘lock’ are believed to play a role in maintaining receptors in inactive states, the lock is observed to be broken in a number of structures of GPCRs bound to inverse agonists and antagonists. This is consistent with the fact that some receptor-T4L fusions exhibit elevated agonist affinities (compared to the native receptors), a characteristic that is associated with enhanced agonist-independent receptor activity of some receptor variants (that do not contain T4L) (Rosenbaum et al., 2007
Understanding of mechanisms of receptor activation would be enhanced by the availability of structures of receptors that are capable of activation of the cognate G-protein. Furthermore, introduction of full-length fluorescent proteins or protein interaction domains could provide tools useful for understanding GPCR action outside of the area of structure determination. To date, the primary approaches for identifying optimal sites of T4L insertion have involved cycles of design and testing of fusion constructs. As an alternative approach for identifying optimal receptor-T4L fusions, we used created a library of receptor variants containing insertions of T4L into all possible positions within a selected region of the third loop of the yeast α-factor receptor Ste2p, accompanied by varying extents of deletion and duplication of loop sequences. Screening of the complete set of receptor-T4L fusions in this library, using screens to identify alleles providing maximal receptor expression, ligand binding and signaling function, led to the identification of receptors with insertions of T4L in the third intracellular loop that exhibit robust ligand-dependent activation of the downstream G protein pathway. The functional fusions we identified all contain duplication of IC3 loop sequences flanking the T4L insertion created via the particular cloning strategy used for introduction of T4L sequences.
Previous studies have implicated the third intracellular loops of various GPCRs in the activation of G proteins (Strader et al., 1994
; Taylor et al., 1994
; Erlenbach and Wess, 1998
; Thompson et al., 1998
; Wess, 1998
). The IC3 loop has also been specifically implicated in the signaling function of the α-factor receptor (Clark et al., 1994
; Stefan and Blumer, 1994
; Celic et al., 2003
). This raises the question of how a receptor can maintain competence to activate G protein with T4L inserted into the critical IC3 region. One rationale for using T4L as a fusion partner is the proximity of its N- and C-termini, which reside <11 Å apart in crystal structures of the isolated enzyme (Rosenbaum et al., 2007
). Since closely packed helices are ~10–15 Å (center to center), properly placed T4L may fit into loops between helices of transmembrane proteins with minimal perturbation of the helical core conformations. (Insertion of T4L into a hydrophilic loop of the lac permease was compatible with retention of lactose transport (Engel et al., 2002a
)). However, even if insertion of T4L into the third intracellular loop of a GPCR does not interfere with the loop's conformation or dynamics, it is surprising that the presence of a ~20 kD protein inserted at this critical position does not sterically interfere with critical receptor-G protein contacts. The simplest explanation of retention of signaling activity by Ste2p variants with the T4L insertions is that the flexible attachment and the extra chain length of the duplicated IC3 sequences in these fusions allow the lysozyme to move out of the way of the critical interactions between IC3 loop sequences and Gα. The role of IC3 flexibility in allowing productive interactions between T4L-containing receptors and G proteins is supported by the observation that all recovered functional T4L-inserted receptors contained extra lengths of IC3 insertion compared with the normal IC3 loop of Ste2p.
Since we find that different regions of IC3 sequences are duplicated in different functional Ste2p-T4L fusion proteins, the retention of native-like interactions with G protein appears to depend more on the presence of extra amino acids than on a role for a specific duplicated sequence. The third intracellular loop of Ste2p has been previously shown to be tolerant of a wide variety of amino acid substitutions (Celic et al., 2003
) and even of a discontinuity of the peptide chain in this region, since fragments of Ste2p comprising the first five predicted transmembrane segments and the last two segments are capable of associating to form an active α-factor receptor (Martin et al., 1999
). Consistent with the current results, successful assembly occurred when the fragments retained intact sequences adjacent to the cytoplasmic ends of TM5 and TM6, but not when the discontinuity was close to the cytoplasmic end of TM5.
Although C-terminal truncation of the cytoplasmic tail of native α-factor receptors results in increased receptor sensitivity to ligand and an increased number of ligand-binding sites at the cell surface (Konopka et al., 1988
; Reneke et al., 1988
), we were unable to recover any tail-less Ste2p-T4L fusions that retained the ability to activate G proteins. Since removing both the receptor's C-terminal tail and C-terminal GFP tag from active full-length Ste2p-T4L fusions resulted in loss of signaling function, the inability of the truncated receptor-T4L fusions to signal must be a direct consequence of the lack of the tail rather than any effect of the fusion to GFP. This suggests that, although it is not directly required for signal transduction, an interaction between the third intracellular loops and the tail may serve to stabilize or promote correct folding or trafficking of altered forms of the receptor. Alternatively, the tail may undergo interactions with the G protein that are not essential for activation of the G protein but may strengthen the interactions with G protein by receptors with altered IC3 loops (Dosil et al., 2000
). The role of the C-terminal tail in formation of the receptor-G protein complex is of particular interest in view of the fact that most of this region has been removed from the receptors for which structures are available from X-ray crystallography or has been found (with the exception of the eighth helix) to be disordered (Choe et al., 2011
Two rationales have been provided for the benefits of using T4L fusions for promoting crystallization of transmembrane proteins. One of these is the reduction in structural heterogeneity and the protection against denaturation that are expected to result from insertion of a stable, soluble protein into a region that is likely to connect two mobile regions of a membrane protein. The other rationale is that the presence of the T4L simply provides additional hydrophilic surfaces that, based on the tendency of T4L to crystallize as an isolated soluble protein, are likely to promote crystallization of the membrane proteins into which it is inserted. The procedures we describe have been used to recover insertions of T4L into third intracellular loops of receptors that maintain strong signaling function. The observation that duplication of IC3 sequences flanking an inserted T4L results in expression of many more cell surface-binding sites than are observed for Ste2p variants containing the duplicated IC3 sequences without T4L raises the possibility that the presence of the lysozyme may actually enhance the receptor's stability in cells. However, the increase in binding sites in the presence of lysozyme could also reflect enhanced folding or intracellular trafficking of T4L-containing receptors.
We do not yet know whether insertion of T4L into signaling-competent receptors will provide stabilization useful for enhancing crystallization. While flexibility of the extended IC3 loop sequences may reduce the rigidity of the T4L-containing receptors, the presence of T4L is expected to provide interacting surfaces that could enhance crystallization. The recent determinations of the structure of the β2-adrenergic receptor-G protein complex using a receptor construct containing T4L fused at its extreme N-terminal (Rasmussen et al., 2011b
) and the structure of the nociceptin/orphanin opioid receptor using a construct containing a stabilized apocytochrome b562
fused at the receptor's N-terminal (Thompson et al., 2012
) suggest that the addition of the interacting surfaces in the absence of receptor stabilization may be sufficient to significantly promote receptor crystallization. We anticipate that the protocols we have developed and that the knowledge that GPCR function can be maintained even when a soluble protein is inserted into the third intracellular loop will be useful in further structural and functional studies of GPCRs.