022P Nottingham, UK
7th Focused Meeting on Cell Signalling

 

 

Systematic mutation reveals different phospho-interaction patterns of beta-arrestin-1 to GPR35 and GPR120

L. Lin, A. Tobin, G. Milligan. Institute of Molecular, Cell and Systems Biology, Glasgow, United Kingdom.

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.