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022P Nottingham, UK 7th Focused Meeting on Cell Signalling |
Systematic mutation reveals different phospho-interaction patterns of beta-arrestin-1 to GPR35 and GPR120
Introduction: Decreased G protein-coupled receptor (GPCR) internalization or highly-activated β-arrestin-biased signalling is closely associated to diseases such as cardiovascular disease or cancer respectively [1,2]. Further investigation of GPCR-β-arrestin interaction may therefore be important for understanding the mechanism(s) involving in the development and progression of these diseases. Currently, the receptor phospho-barcode and how this modulates the interaction pattern with β-arrestins are poorly understood. In a previous report, seven phosphate-binding sites of β-arrestin-1 were identified by an NMR approach [3]. Here, we have generated seven β-arrestin-1 mutants corresponding to these sites and demonstrate: 1) GPR35 and GPR120 interact with different phosphate-binding sites of β-arrestin-1; 2) some mutations of β-arrestin-1 lead to greater levels of GPCR-β-arrestin-1 interaction.
Methods: GPR35 and GPR120 receptor constructs contained enhanced yellow fluorescent protein (eYFP) fused to the C terminus as previously described [4,5]. Constructs of wild type and mutant bovine β-arrestin-1 with NanoLuc luciferase fused to their N terminus were generated and applied in bioluminescence resonance energy transfer (BRET) assays. β-arrestin-1 mutations include site1 (Y63A/R65A/K77A), site2 (K160A/R65A/R165A), site3 (K160A/K11A), site4 (R25A), site5 (R7A), site6 (R7A), and site6/7 (Y21A/K107A/K10E). Agonists applied here were zaprinast for GPR35 and TUG-891 for GPR120. Results represent the mean±SEM.
Results: Results are shown as Table 1:
1) GPR35 interacts with sites 1, 2, 3, 4 of β-arrestin-1, whereas GPR120 interacts with sites 2, 3, 6.
2) Site 7 displays species selective β-arrestin-1 interactions between mouse and human GPR35
3) Site 6 or sites 1, 5 significantly increase β-arrestin-1 interactions with GPR35 or GPR120 respectively.
Table 1. β-arrestin-1 recruitment (% max WT response).
WT | SITE1 | SITE2 | SITE3 | SITE4 | SITE5 | SITE6 | SITE6/7 | |
---|---|---|---|---|---|---|---|---|
h35 | 100.0±5.0 | 33.8±3.6 | 46.0±5.0 | 15.8±4.0 | 59.7±2.7 | 100.7±1.5 | 140.0±1.4 | 196.4±7.7 |
m35 | 100.0±3.1 | 53.8±7.8 | 51.3±4.2 | 17.7±2.5 | 29.0±2.8 | 109.7±4.0 | 188.5±3.4 | 69.9±3.0 |
h120 | 100.0±3.4 | 177.8±0.3 | 66.3±0.3 | 61.4±2.8 | 126.7±6.4 | 200.1±4.7 | 73.9±1.9 | 106.6±7.5 |
m120 | 100.0±0.2 | 158.9±1.4 | 52.1±2.9 | 47.7±2.3 | 113.7±0.9 | 137.4±0.8 | 78.8±2.6 | 123.3±5.2 |
Conclusions: β-arrestin-1 displays different phospho-interaction patterns with GPR35 and GPR120. These differences may allow β-arrestin-1 to activate different downstream targets and contribute to the functional diversity of GPCR receptors in their control of physiological responses.
References:
1. Carr et al. (2016) Proc Natl Acad Sci U S A. 113:E4107-E4116.
2. Kose et al. (2017) Bioorganic & Medicinal Chemistry Letters. 27:3611-3620.
3. Yang et al. (2015) Nat Commun. 6:8202.
4. Jenkins et al. (2011) Br J Pharmacol. 162:733-748.
5. Hudson et al. (2013) Mol Pharmacol. 84:710-725.