archives@tulane.edu / G protein-coupled receptors (GPCRs) constitute the largest family of human proteins with approximately around 800 genes and, are therapeutic targets for more than 35% of the FDA-approved drugs with around 180 billion dollars in sales. The therapeutic potential of GPCRs is still not completely utilized. Structure-guided drug discovery will definitely aid in identifying novel compounds with therapeutic responses while minimizing adverse effects in patients. Multiple protein engineering strategies including deletions, truncations, insertion of stable soluble protein or thermostabilizing mutations have been utilized to obtain the structural information of these membrane proteins. Yet to date, the high-resolution structure of only 63 GPCRs have been determined, with only a few of these structures being of active protein. Heterologous expression of most GPCRs results in low yields of active receptors and additional strategies are required to improve active receptor yields for these “hard-to-express” GPCRs. Yeast provides a unique heterologous expression platform to produce mammalian GPCRs, as it also allows us to measure downstream signaling activity with an easy and inexpensive high-throughput assay via its MAPK response pathway.
The adenosine A2A receptor (A2AR) shows exceptional expression and trafficking to the plasma membrane in yeast; however, this is not the case for other adenosine receptors. A2AR has a longer C-terminus than the other adenosine receptor subtypes, which may contribute to its exceptional trafficking to the plasma membrane. To test the possibility to improve trafficking of the adenosine A1 receptor (A1R), chimeric receptors containing the seven transmembrane domains of A1R and the full-length or truncated A2AR C-terminus were constructed. The chimeric receptor showed improved localization to the plasma membrane and was capable of binding radioligand with native-like A1R affinity. Functionally active A1R receptor variants were produced at a theoretical yield of 95 pmol/mg total membrane protein, estimated using radioligand binding data, which is greater than three-fold higher than previously reported yields from other heterologous expression systems, and should facilitate biophysical characterization and drug discovery efforts.
Recent efforts to determine the high-resolution crystal structures for the adenosine A1 and A2A receptors have utilized modifications to the native receptors in order to facilitate receptor crystallization and structure determination. One common modification is a truncation of the unstructured C-terminus, which has been employed for all the adenosine crystal structures obtained to date. Here, the presence of the full-length C-terminus is shown to affect downstream signaling using a yeast MAPK response-based fluorescence assay. Upon ligand binding, the A1d291R or A2Ad316R variants were unable to couple to human-yeast chimeric G-protein chimeras to generate a downstream signal in yeast, though full-length receptors showed native-like G-protein coupling. Further, constructs transfected into HEK-293 cells showed similar behavior – i.e. the variants with C-terminal truncations lacked cAMP-linked signaling compared to the full-length receptors. Although the C-terminus was essential for Ga protein- associated signaling, chimeras of A1R with a C-terminus of A2AR coupled to the A1R-specific Ga (i.e. Gai1 versus Gas). This surprising result suggests that the C-terminus is important in signaling, but not specificity, for the interaction with Ga protein.
Based on these results for chimeric A1R variants, chimeric variants for adenosine A3R and tachykinin 2 receptor (NK2R) were constructed. The A3/A2AR variant was constructed using an approach similar to that for A1/A2AR; the resulting construct showed a two-fold increased expression as compared to the wild-type A3R. This chimeric variant showed native-like signaling activity in engineered yeast strains with a modified MAPK pathway, whereas the wild-type receptor showed no activity.
In order to improve the functional expression of human NK2R required for high-resolution structural studies, NK2R chimeras utilizing sequences from rat NK2R previously shown to have a higher functional expression in yeast than human NK2R were utilized. Herein, we show that NK2R chimeras incorporating the rat NK2R C-terminus demonstrated improved expression, ligand binding, and downstream signaling in engineered yeast strains. Taken together, these results highlight the utility of the chimeric receptor engineering strategy to facilitate the expression of active receptors. / 0 / Abhinav Rabindra Jain
| Identifer | oai:union.ndltd.org:TULANE/oai:http://digitallibrary.tulane.edu/:tulane_108966 |
| Date | January 2019 |
| Contributors | Jain, Abhinav Rabindra (author), Robinson, Anne S (Thesis advisor), School of Science & Engineering Chemical and Biomolecular Engineering (Degree granting institution) |
| Publisher | Tulane University |
| Source Sets | Tulane University |
| Language | English |
| Detected Language | English |
| Type | Text |
| Format | electronic, pages: 207 |
| Rights | 12 months, Copyright is in accordance with U.S. Copyright law. |
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