Transferability of thermostabilizing mutations between β-adrenergic receptors

Figures & data

Figure 1.  (a) Sequence alignment between the turkey β₂AR (adrb_melga) and human βARs (adrb1/2/3_human). The N- and C-termini of the sequences were removed in the alignment, because there is no significant homology between them. Amino acid residues coloured red in the turkey sequence correspond with mutations present in tβ₂AR-m23 (R68S, M90V, Y227A, A282L, F327A and F338M), whereas those coloured in blue correspond with other thermostabilizing mutations. Letters above these residues correspond to the position of the residues in (b). Pink rectangles below the sequence alignment indicate the positions of transmembrane domains and the green rectangle corresponds with the position of helix 8. (b) Structure of the turkey β₂AR (PDB accession number 2VT4) with the side chains of thermostabilizing mutations represented as space filling models. Side chains in grey are thermostabilizing when mutated to alanine. The five magenta side chains correspond to the five thermostabilizing mutations found in tβ₂AR-m23, i.e., R68S, M90V, Y227A, F327A and F338M; the mutation A282L is not shown because this region of the receptor was disordered in this crystal form. The N- and C-termini are labelled, along with the transmembrane α-helices. The model is in rainbow coloration with the N terminus in blue and the C terminus in red. The black sphere is a Na⁺ ion. This Figure is reproduced in colour in Molecular Membrane Biology online.

Figure 2.  Expression and binding assays of turkey β₂AR and human βARs expressed in HEK293 cells. The number of receptors per cell was calculated from single-point ligand binding assays using [³H]DHA at a final concentration of 80 nM performed either on membranes (a) or on cells solubilized with 2% DDM (b). Cells were transfected either with empty vector (V), turkey β₂AR (tβ₂), human β₂AR_1-463 (hβ₂) or human β₂AR (hβ₂). Experiments were performed in duplicate and the error bars represent the standard error of the mean (SEM). This Figure is reproduced in colour in Molecular Membrane Biology online.

Figure 3.  The effect of increasing detergent concentrations on the [³H]DHA binding activity of βARs. Receptors were transiently expressed in HEK293 cells, which were subsequently solubilized at 4°C in different concentrations of DDM (0.01%, 0.05%, 0.1%, 0.5% and 1%); the amount of solubilized functional receptor was determined from a single-point binding assay performed at 4°C using 80 nM [³H]DHA; (a) turkey β₂AR, (b) human β₂AR_1-463 and (c) human β₂AR. Experiments were performed in duplicate and the error bars represent the SEM.

Figure 4.  The stability of βARs with and without thermostabilizing mutations. Receptors were solubilized from transiently transfected cells using either 0.1% (red squares) or 0.01% DDM (blue triangles). Samples were then heated at the specified temperature for 30 minutes, quenched on ice and the amount of receptor remaining determined by a single-point ligand binding assay using 80 nM [³H]DHA performed in duplicate; (a) turkey β₂AR, (b) human β₂AR_1-463, (c) human β₂AR, (d) turkey β₂AR-m23, (e) human β₂AR_1-463-m23, (f) human β₂AR-m23. Stability curves were analyzed by non-linear regression using Prism (GraphPad) to determine the T_ms (Table I). This Figure is reproduced in colour in Molecular Membrane Biology online.

Table I.  Apparent T_ms of β receptors solubilized in different concentrations of dodecylmaltoside (DDM) and decylmaltoside (DM).

	DDM		DM
Receptor	[DDM](%)	Apparent Tm (°C)	[DM](%)	Apparent Tm (°C)
Turkey β1AR	0.01	31
	0.1	23
Turkey β1AR-m23	0.01	45
	0.1	45
Human β1AR	0.01	21
	0.1	10	0.1	10
Human β1AR-m23	0.01	38
	0.1	27	0.1	20
Human β2AR	0.01	30
	0.1	29	0.1	22
Human β2AR-m23	0.01	41
	0.1	41	0.1	26

Table II.  Affinity of agonist and antagonist binding to detergent solubilized βARs.

Receptor	Ki isoprenaline (μM)*	Ki adrenaline (μM)*	Ki cyanopindolol (nM)*
Human β1AR	0.08 ± 0.01	0.52 ± 0.14	6.4 ± 0.7
Human β1AR-m23	14 ± 2	190 ±50	3.8 ± 0.1
Human β2AR	21 ± 3	7.1 ± 1.3	3.5 ± 0.1
Human β2AR-m23	94 ± 11	1440 ± 110	2.8 ± 0.2

*K_is were calculated using the Cheng & Prusoff equation from IC50s determined from competition assays (Figure 6) that contained [³H]DHA at a concentration three times the K_D (Supplementary Table S3, online version only). Errors are the SEM from the data fitting to the competition curves.

Figure 5.  Resistance of the human β₂AR_1-463 and β₂AR to decylmaltoside. (a) Receptors were solubilized with different amounts of decylmaltoside (DM) and the ligand binding activity measured using 80 nM [³H]DHA. Red bars, hβ₂AR_1-463; pink bars, hβ₂AR_1-463-m23; dark blue bars, hβ₂AR and light blue bars, hβ₂AR-m23. The DM concentrations used are as follows: a, 0.1%; b, 0.3%; c, 1%. (b) The stability of the hβ₂AR_1-463 and hβ₂AR solubilized in decylmaltoside (DM) was determined by incubating the sample for 30 minutes at different temperatures, quenching on ice and determining the amount of remaining receptors by assaying with 80 nM [³H]DHA: red, hβ₂AR_1-463; pink, hβ₂AR_1-463-m23; dark blue, hβ₂AR and light blue, hβ₂AR-m23. Stability curves were analysed by non-linear regression using Prism (GraphPad) to determine the T_ms (Table I). This Figure is reproduced in colour in Molecular Membrane Biology online.

Figure 6.  Competition binding assays of human βARs with and without the m23 thermostabilizing mutations. Increasing amounts of the agonists, isoprenaline (a) and adrenaline (b), and the antagonist cyanopindolol (c), were used to compete with binding of [³H]DHA present at a concentration three times greater than the K_D for each receptor (Supplementary Table S3, online version only): red squares, hβ₂AR_1-463; pink circles, hβ₂AR_1-463-m23; dark blue triangles, hβ₂AR; pale blue inverted triangles, hβ₂AR-m23. Data were analyzed with GraphPad Prism and the K_is determined (Table II). This Figure is reproduced in colour in Molecular Membrane Biology online.