Functional fusions of T4 lysozyme in the third intracellular loop of a G protein-coupled receptor identified by a random screening approach in yeast

Abstract

The insertion of a stable soluble protein into loops of transmembrane proteins has proved to be a successful approach for enhancing their stabilities and crystallization, and may also be useful in contexts where the inserted proteins can modulate or report on the activities of membrane proteins. While the use of T4 lysozyme to replace portions of the third intracellular loops of G protein-coupled receptors (GPCRs) has allowed determination of the structures of members of this important class of receptors, the creation of such fusion proteins generally leads to loss of signaling function of the resulting fusion protein, since the third intracellular loops of GPCRs play critical roles in their interactions with G proteins. We describe here a random screening approach allowing insertion of T4 lysozyme into diverse positions in the third loop of the yeast α-pheromone receptor, a GPCR encoded by the yeast STE2 gene. Insertions were accompanied by varying extents of deletion or duplication of the loop. A set of phenotypic screens allow detection of potentially rare variant receptors that are expressed, bind to agonist and are capable of signal transduction via activation of the cognate G protein. A large fraction of screened full-length receptor variants containing at least partial duplications of the loop on either side of the inserted T4 lysozyme retain the ability to activate the downstream signaling pathway in response to binding of ligand. However, we were unable to identify any receptors with truncated C-termini that retain significant signaling function in the presence of inserted T4 lysozyme. Our results establish the feasibility of creating functional receptors containing insertions of T4 lysozyme in their third intracellular loops.

A Novel Screening Approach for Optimal and Functional Fusion of T4 Lysozyme in GPCRs

“…Application of the described procedures for creating a library of variant forms of the yeast α-factor receptor containing T4L inserted into the IC3 loop allowed efficient recovery of the full range of possible receptor variants, with some bias against variants containing insertions with the shortest loop sequences (Mathew, et al, 2013). Based on GFP fluorescence, many of the variant receptors were expressed at levels that were similar to the levels of receptors with no insertions.…”

“…While random screening uncovered some C-terminally truncated Ste2p-T4L fusions that bound up to 18% of the high levels of ligand bound by similarly-expressed truncated receptors with no insertion, none of the truncated receptors containing the T4L indertion exhibited more than ~5% of the signaling activity of the unfused receptors. The C-terminal tail may, therefore, provide stabilization to receptors containing perturbations such as inserted proteins (Mathew, et al, 2013). This is an important point to be considered in view of the fact that GPCR constructs used for crystallography involve truncations of the C-terminal to remove regions presumed to have excess flexibility.…”

“…pMD1859 – Created by site directed mutagenesis of plasmid pMD1167, replacing the IC3 loop of STE2 with nucleotide sequences that code for two BsaBI restriction sites flanking a SmaI site (confers ampicillin-resistance in E.coli and uracil auxotrophy in S.cerevisiae ) (Mathew, et al, 2013). …”

“…We describe here a strategy for generating and screening a library of variant forms of a receptor containing insertions of T4L at different points in the third intracellular loop (IC3) as a replacement for different numbers of amino acid residues in the loop sequence (Mathew, et al, 2013). By expressing receptors in yeast, it is possible to create randomized libraries of variants with lysozyme insertions, and to directly screen for constructs with maximal overall expression levels, numbers of ligand binding sites at the cell surface, and even receptor function, assayed based on activation of the cognate G protein.…”

Fluorescent approaches for understanding interactions of ligands with G protein coupled receptors

“…However, the approach has also been used for ligands for adenosine [85, 86], adrenergic [87, 88], cannabinoid [89], chemokine [90, 91], galanin [92], long chain fatty acid [93], neuropeptide Y [94], somatostatin [95, 96], urotensin [97], vasopressin [98]and yeast α-factor [82, 99–102] receptors.…”