Specific cell surface labeling of GPCRs using split GFP

Abstract

Specific cell surface labeling is essential for visualizing the internalization processes of G-protein coupled receptors (GPCRs) and for gaining mechanistic insight of GPCR functions. Here we present a rapid, specific, and versatile labeling scheme for GPCRs at living-cell membrane with the use of a split green fluorescent protein (GFP). Demonstrated with two GPCRs, GPR17 and CysLT2R, we show that two β-stands (β-stands 10 and 11) derived from a superfolder GFP (sfGFP) can be engineered to one of the three extracellular loop of a GPCR. The complementary fragment of sfGFP has nine β-strands (β-stands 1-9) that carries the mature fluorophore, and can be proteolytically derived from the full-length sfGFP. Separately the GFP fragments are non-fluorescent, but become fluorescent upon assembly, thus allowing specific labeling of the target proteins. The two GFP fragments rapidly assemble and the resulting complex is extremely tight under non-denaturing conditions, which allows real-time and quantitative assessment of the internalized GPCRs. We envision that this labeling scheme will be of great use for labeling other membrane proteins in various biological and pharmacological applications.

Figure 1. Illustration of the split GFP scheme for the cell surface labeling of a GPCR protein.

(A) GFP structure, with GFP β-strands 1-9 (GFP1-9) colored in green and GFP β-strands 10-11 (GFP10-11) colored in gray. A trypsin cleavage site was introduced between the 9^th and 10^th β-strands, as indicated. (B) Schematic diagram for the preparation of GFP1-9. Following trypsin digestion, the two fragments were separated in the presence of 3 M guanidine hydrochloride. GFP1-9 was then purified and refolded. (C) Absorption and fluorescence emission spectra for wild type GFP, GFP1-9, and GFP10-11:GFP1-9 complex. (D) Schematic diagram for the assembly between GFP1-9 and GFP10-11 engineered to the third extracellular loop of GPR17, a GPCR protein.

Figure 2. The assembly between GFP1-9 and GFP10-11 in solution.

(A) Schematic diagram showing the assembly between GFP1-9 and GFP10-11 engineered to the first loop of ubiquitin. (B) SDS PAGE gel analysis of GFP1-9, Ub/GFP10-11, and the complex between GFP1-9 and Ub/GFP10-11. (C,D) Stoichiometric binding between GFP1-9 and Ub/GFP10-11. At non-saturating concentrations, the fluorescence intensity increased linearly with the increase of Ub/GFP10-11 protein concentration. (E,F) Kinetics for the assembly between 1000 nM GFP1-9 and 1000 nM Ub/GFP10-11. Fluorescence signal intensity plateaus in less than 5 min.

Figure 3. Surface labeling of GPR17 in HEK293 cells.

(A) Representative images of the cells transfected with wild type GPR17 (GFP17/WT), GPR17/R214/GFP10-11, or GPR17/R291/GFP10-11, in the presence or absence of GFP1-9. (B) Kinetics of the assembly between GFP1-9 and GPR17/R291/GFP10-11 in living cells, characterized by the increasing green fluorescence signal at the cell surface. (C) Flow cytometry measurements of GPR17. The fluorescence intensity at 488 nm was quantified for cells transfected with GPR17/WT, GPR17/R291/GFP10-11, or GPR17/R214/GFP10-11 in the absence or presence of GFP1-9.

Figure 4. Visualization the internalization of GPR17.

(A) Representative images of the split GFP labeled HEK293 cells at 0 h, 12 h and 24 h in the absence or presence of UDP, a GPR17 ligand. (B) Representative images of the split GFP labeled living cells before and 30 s after the administration of 2 M NaI. The NaI was applied either right after or 24 h after split GFP labeling. (C) The statistical analysis of GPR17 internalization for cells with split GFP labeling. ***P < 0.001, compared with 0 h after split GFP labeling with no UDP, one-way ANOVA.