Characterization of binding kinetics of A2AR to Gαs protein by surface plasmon resonance

Abstract

Because of their surface localization, G protein-coupled receptors (GPCRs) are often pharmaceutical targets as they respond to a variety of extracellular stimuli (e.g., light, hormones, small molecules) that may activate or inhibit a downstream signaling response. The adenosine A_2A receptor (A_2AR) is a well-characterized GPCR that is expressed widely throughout the human body, with over 10 crystal structures determined. Truncation of the A_2AR C-terminus is necessary for crystallization as this portion of the receptor is long and unstructured; however, previous work suggests shortening of the A_2AR C-terminus from 412 to 316 amino acids (A_2AΔ316R) ablates downstream signaling, as measured by cAMP production, to below that of constitutive full-length A_2AR levels. As cAMP production is downstream of the first activation event-coupling of G protein to its receptor-investigating that first step in activation is important in understanding how the truncation effects native GPCR function. Here, using purified receptor and Gα_s proteins, we characterize the association of A_2AR and A_2AΔ316R to Gα_s with and without GDP or GTPγs using surface plasmon resonance (SPR). Gα_s affinity for A_2AR was greatest for apo-Gα_s, moderately affected in the presence of GDP and nearly completely ablated by the addition of GTPγs. Truncation of the A_2AR C-terminus (A_2AΔ316R) decreased the affinity of the unliganded receptor for Gα_s by ∼20%, suggesting small changes to binding can greatly impact downstream signaling.

Figure 1

Typical results from G protein and receptor purification for protein purity and expression quantification. (A) Coomassie stain shows improvement in purity for various steps throughout Gα_s purification, with less than 6% present in the crude lysate. (B) Anti-His western blot to detect Gα_s present at various steps throughout purification. Molecular weight markers (Precision Plus Protein WesternC Standards; Bio-Rad Laboratories) were used to identify protein migration, as shown in all samples. For both (A) and (B), Lane L1 represents whole cell lysate, L2 soluble protein lysate (post centrifuge), and L3 lysate from flowthrough of Ni-NTA resin. FT1 and FT2 represent flow through after wash 1 and 2, respectively, whereas eluted fractions (E1–E5) and the pooled combined fractions (P) are indicated. Arrow indicates Gα_s at 46 kDa. (C) SYPRO Ruby-stained membrane and (D) anti-A_2AR Western blot of purified 1) A_2AR-1d4 2), A_2AΔ316R-1d4, and 3) A_2AR-His₁₀ showing similar protein purities between purification strategies and receptors. A_2AR bands are present, as indicated. Note that the mobilities are slightly faster than that expected by calculated molecular weight (45 and 37 kDa); membrane proteins have been found to run faster than expected, possibly because of their hydrophobicity or incomplete denaturation due to the absence of sample heating.

Figure 2

A_2AR shows specific binding to Gα_s, as determined by SPR. (A) Sensorgram of a typical experiment from start (Ni²⁺ injection) to finish (EDTA regeneration). Buffer, Ni²⁺, A_2AR, and EDTA flow simultaneously over channel 1 (gray dotted line) and channel 2 (gray solid line). Denatured Gα_s and active Gα_s are only injected on channels 1 and 2, respectively. Specific binding (channel 2 to channel 1) is shown as a solid black line to demonstrate the calculation performed for each experiment. (B) Injection of 300 nM purified Gα_s at time = 0 s. Injection lasts for 60 s, after which a new baseline is established from bound protein. Data are from one flow channel of one experiment as a representative indicator of Gα_s associating with Ni-NTA chip. (C) 2000 nM purified A_2AR is injected at time = 60 s at 10 μL/s across denatured Gα_s (gray dotted line) and active Gα_s (gray solid line). Nonspecific binding of A_2AR to denatured Gα_s is subtracted from that of A_2AR to the active Gα_s curve to give a specific binding curve (black solid line) for each multichannel SPR run. Baseline sensorgram curve was set to 0 RU to compare between sensorgrams from different flow paths. Data are representative of typical results from a single experiment and expressed in RU.

Figure 3

Association of A_2AR to Gα_s affected by the addition of GDP and GTPγs. 2000 nM unliganded A_2AR was injected at time zero onto Gα_s preincubated with buffer alone (solid black line), GDP (dashed medium gray line), or Gα_s-GTPγs (dotted light grey line). Data fits were performed for specific binding of six concentrations of A_2AR (263–2000 nM) from three independent purifications to obtain kinetic constants shown in Table 1, but for clarity, only 2000 nM data for one purification run as a technical duplicate are shown in this figure. In this figure, although all samples were run in duplicate (n = 2), a single line may appear because of the small noise relative to the signal.

Figure 4

Specific binding of A_2AR and A_2AΔ316R to Gα_s shows concentration and C-terminus dependence. Sensorgrams show average specific binding (n = 6) of purified (A) A_2AR or (B) A_2AΔ316R at 263, 395, 592, 889, 1333, and 2000 nM (arrows indicate increasing receptor concentrations) for Gα_s attached to Ni-NTA chip. Kinetic constants were determined (Table 1) based on a global fit of the data. Error bars are omitted from the figure for clarity of presentation.