2.2 Methods
3.2.2 Arrestin recruitment to the β 1 -adrenoceptor
It is believed that intracellular phosphorylation is a prerequisite for arrestin binding to G protein-coupled receptors. Therefore we investigated arrestin binding to the β1-adrenoceptor using a FRET-based arrestin recruitment assay as described in chapter1.4.3.
To verify accurate membrane and intracellular localization of the Cer-taggedβ1 -adrenoceptor and the YFP-taggedβ-arrestin2, respectively, a transient transfection following confocal microscopy was performed. Figure3.13depicts the membrane localization of serveral ADRB1-Cer variants and the cytosolic localization of YFP-β-arrestin2.
Impact of the third intracellular loop on arrestin recruitment
The strongest phosphorylation regulation, that was observed in the SILAC exper-iment (Chapter 3.1.3), was in the third intracellular loop, namely at serine 260.
We therefore investigated the impact of phosphorylation in the third intracellular loop on arrestin recruitment to the ADRB1 using two ADRB1 mutants to cover the potential phosphorylation sites. The first mutant, ADRB1∆3rdloopa, was a single site mutant: only Ser260 was mutated to Ala260. The second receptor variant, ADRB1∆3rdloopb, contained five potential phosphorylation sites: Ser274, Ser276, Ser278, Thr298 and Ser312. These five residues had all been mutated to alanine (Figure3.14(A)).
Figure 3.13:Membrane localization of differentβ1-adrenoceptor mutants.
Confocal microscopy of different cerulean-taggedβ1-adrenoceptor mutants and YFP-tagged β-arrestin2. The receptors are correctly located in the membrane, while β-arrestin2 exhibits a cytosolic expression pattern.
The different receptor variants were all Cer-tagged and β-arrestin2 was N-terminally fused to YFP. When the two fluorophores were in close proximity, this led to an increase in FRET ratio. The completely phoshodeficientβ1-adrenoceptor ex-hibited a significantly lower FRET ratio compared to wild-type (Figure3.14(B) and
(C)). However, neither ADRB1∆3rdloopanor ADRB1∆3rdloopb showed any change inβ-arrestin recruitment as compared to wild-type ADRB1 (Figure3.14(C)).
Figure 3.14:Phosphorylation sites in the third intracellular loop do not influence arrestin re-cruitment to the ADRB1.
(A) β1-adrenoceptor schematic highlighting the phosphosite mutants of the third intracellular loop. Light green: ADRB1∆3rdloopa, dark green: ADRB1∆3rdloopb (B) Mean ±SEM of 10-12 representative FRET tracings comparing wild-type ADRB1, ADRB1∆3rdloopaand ADRB1∆phos. In ADRB1∆phos all serine and threonine residues in the third intracellular loop and in the C-terminus were mutated to alanine residues.
(C) Quantification of β-arrestin2 recruitment to different ADRB1 variants. Mean + SEM of 44-74 FRET amplitudes. In ADRB1∆3rdloopball serines and threonines in the third intracellular loop except for Ser260 have been mutated to alanine residues.
Kruskal-Wallis-Test with Dunn‘s post test. **** p≤0.0001 vs. wild-type and n.s. = not significant.
Impact of the C-terminus on arrestin recruitment
To elucidate, where the significant decrease inβ-arrestin2 recruitment seen with the ADRB1∆phos variant (Figure 3.14 (B) and (C)) originated, the C-terminal phosphorylation sites of theβ1-adrenoceptor were investigated. The large size of the C-terminus made it favorable to cover it by three different phosphodeficient β1-adrenoceptor mutants, as opposed to two in case of the third intracellular loop.
ADRB1∆proximal covered two serines and one threonine residue. Thr404, Ser412 and Ser423 had been exchanged for alanine in this receptor mutant. For the ADRB1 mutant ADRB1∆middle, two amino acids were mutated to alanine: Ser428 and Thr439. The last ADRB1 variant, ADRB1∆distal, spanned five serine residues:
Ser459, Ser461, Ser462, Ser473 and Ser475 (Figure3.15(A)).
Both ADRB1∆proximal as well as ADRB1∆distal showed an impairment in β-arrestin2 recruitment to theβ1-adrenoceptor (Figure3.15(B)). For the∆distal mu-tant the effect was comparable to the reduction inβ-arrestin2 recruitment seen with the completely phosphodeficient ADRB1 mutant∆phos. ADRB1∆middle showed no change in arrestin recruitment as compared to wild-type ADRB1 (Figure3.15 (C)).
Figure 3.15:Phosphorylation sites in the C-terminus are crucial forβ-arrestin2 recruitment.
(A) Schematic of the C-terminally phosphodeficient ADRB1 variants: ADRB1∆proximal (dark grey),∆middle (ivory) and∆distal (blue) C-terminus. (B) Mean±SEM of 10-15 FRET tracings comparing wild-type ADRB1, ADRB1∆phos, ADRB1∆proximal and
∆distal C-terminus. (C) Quantification ofβ-arrestin2 recruitment of different ADRB1 variants. Mean + SEM of 30-78 FRET tracing amplitudes. Kruskal-Wallis-Test with Dunn‘s post test. **** p≤0.0001, *** p≤0.001 vs. wild-type and n.s. = not significant.
These results led to a thorough investigation of the distal C-terminus of the β1-adrenoceptor.
Another three mutants were created to take a closer look at the very end of the receptor: Ala459/461/462, Ala461/462 and Ala473/475 (Figure3.16(A)). The two mutants Ala459/461/462 and Ala461/462 both exhibited a significant decrease inβ-arrestin2 recruitment to the receptor variants, while the Ala473/475 mutant showed no change as compared to wild-type ADRB1 (Figure3.16(B) and (C)).
Ser461/462, which are important forβ-arrestin2 recruitment to the ADRB1 show a strong conservation among different vertebrate species. This conservation seems similar to the conservation of the PDZ type I domain, which is located at the very end of the receptor and includes the two serine residues 473 and 475 (Figure3.17).
Figure 3.16:Phosphorylation at serine 461 / serine 462 in the C-terminus determines arrestin binding.
(A) Schematic of the ADRB1 variants representing the distal C-terminus:
ADRB1(Ala459/461/462) (light blue), ADRB1(Ala461/462) (orange) and ADRB1 (Ala473/475) (dark grey). (B) Mean ± SEM of 6-13 FRET tracings comparing ADRB1(Ala459/461/462), ADRB1(Ala461/462) and ADRB1(Ala473/475). (C) Quan-tification ofβ-arrestin2 recruitment to ADRB1 variants. Mean + SEM of 9-36 FRET tracing amplitudes. Kruskal-Wallis test with Dunn‘s post test. *** p≤0.001 vs. wild-type, **** p≤0.0001 and n.s. = not significant.
Figure 3.17:The ADRB1 arrestin binding site is highly conserved among vertebrates.
Positions 459 (light blue), 461 and 462 (orange) are compared among 12 different vertebrate species (human, mouse, zebrafish, duck, cow, guinea pig, dog, squirrel, elephant, turkey, gorilla and chicken). This image was created with Weblogo (Crooks et al., 2004).