Functional competence of a partially engaged GPCR–β-arrestin complex

G Protein-coupled receptors (GPCRs) constitute the largest family of cell surface receptors and drug targets. GPCR signalling and desensitization is critically regulated by β-arrestins (βarr). GPCR–βarr interaction is biphasic where the phosphorylated carboxyl terminus of GPCRs docks to the N-domain of βarr first and then seven transmembrane core of the receptor engages with βarr. It is currently unknown whether fully engaged GPCR–βarr complex is essential for functional outcomes or partially engaged complex can also be functionally competent. Here we assemble partially and fully engaged complexes of a chimeric β2V2R with βarr1, and discover that the core interaction is dispensable for receptor endocytosis, ERK MAP kinase binding and activation. Furthermore, we observe that carvedilol, a βarr biased ligand, does not promote detectable engagement between βarr1 and the receptor core. These findings uncover a previously unknown aspect of GPCR-βarr interaction and provide novel insights into GPCR signalling and regulatory paradigms.


Supplementary Figure 2. Assembly of β2V2R-βarr1-Fab 30 complex. A.
Increasing concentrations of purified Fab 30 were immobilized on to polystyrene surface and maximal immobilization was assessed by reactivity of HRP-coupled Protein L. B. β2V2R and GRK2 were co-expressed in Sf9 or HEK-293 cells followed by stimulation using indicated ligands. C. β2V2R-β-arr1-Fab30 complex assembly requires receptor phosphorylation as agonist bound but dephosphorylated receptor (treated with λ-phosphatase , λ-Phos.) fails to yield a detectable complex formation. D. β2V2R-β-arr1-Fab30 complex assembly is saturable and highly dependent on agonist stimulation (for receptor phosphorylatuon) of the receptor. E. In-vitro assembly of β2V2R-β-arr1 complex depends on Fab 30. A control Fab (Fab CTL) that does not interact with β-arrestin 1 fails to facilitate the assembly of β2V2R-β-arr1 complex. F. Sequence comparison of the carboxyl-terminus of β2AR and β2V2R (blue). Potential phosphorylation sites in β2V2R are underlined and known phosphorylation sites in β2AR are in bold and underlined. Data in panels C and E represent mean±SEM of three independent experiments analyzed by ONE-WAY ANOVA with Bonferroni post-test (***p<0.001).

Supplementary Figure 3. Purification of β2V2R and β2V2R ΔICL3 using baculovirus infected Sf9 cells. A.
Cultured Sf9 cells were infected with baculovirus encoding N-terminal FLAG tagged β2V2R (or β2V2R ΔICL3 ) and untagged GRK2 CAAX . 60-66h post-infection, cells were stimulated with isoproterenol (1μM final at 37°C for 1h), solubilized using 0.5 % LMNG (2h at room-temperature) and purified by anti-FLAG affinity chromatography. Purified receptor was concentrated using a 30kDa cut-off viva-spin concentrator and the purity was analyzed by 10% SDS-PAGE stained with SimplyBlue (Invitrogen). B. Purification table sowing typical purification yields of β2V2R and β2V2R ΔICL3 as measured by total protein estimation (Bradford assay) and gel-based quantification using BSA as standard.

Supplementary Figure 4. Assembly of β2V2R-βarr1-Fab 30 complex with ligands of different efficacies.
A. Incubation of β2V2R ligands with pre-formed Apo β2V2R phos -β-arr1-Fab30 complex followed by coimmunoprecipitation reveals comparable levels of physical interaction. B. Quantification of data presented in panel A. C. Incubation of β2V2R ligands with pre-formed Apo β2V2R phos -β-arr1-Fab30 complex in ELISA format further confirms the comparable levels of physical interaction. D. Addition of varying dosage of full agonist BI-167107 leads to a pattern of bimane fluorescence quenching that directly corresponds to the agonist occupancy of the receptor. Data in panel represents mean ± SEM of three independent experiments. Figure 5. Interaction of βarr1 with ERK2 in presence and absence of V2Rpp. 2.5μg of purified ERK2 (A) or phosphorylated ERK2 (B) was immobilized in each well of a 96 well plate, followed by blocking of non-specific binding sites with 200μl of BSA (0.5%) per well. Subsequently, the wells were incubated with purified and biotinylated βarr1 (2.5μg) in presence or absence of V2Rpp (10 fold molar excess). Post-incubation, wells were washed and incubated with HRP-coupled streptavidin followed by visualization of the signal using TMB ELISA. Data represent mean ± SEM of three independent experiments each carried out in duplicates.

Supplementary Figure 6. Binding of β2V2R-βarr1-ScFv30 complexes with ERK2 MAP kinase. A.
Conversion of Fab30 in to ScFv30 (single chain variable fragment) by joining the coding regions of variable light chain and variable heavy chain through a flexible linker. B. Purified ScFv30 also supports invitro assembly of β2V2R-βarr1 complex as assessed by ELISA. C. ERK2 MAP kinase was purified from E. coli and phosphorylated in-vitro using MEK1 as described in the Materials and Methods section. Phosphorylation status of active ERK2 was probed with anti-phospho-ERK2 antibody and anti-ERK2 antibody. D. Interaction of inactive ERK2 and E. active ERK2 with β2V2R-β-arr1-ScFv30 as assessed by coimmunoprecipitation assay (representative image from three independent experiments). Quantification of complex interaction with F. ERK2 and G. pERK2. Data in panel B represent mean±SEM of three independent experiments (ONE-WAY ANOVA with Bonferroni post-test; ***p<0.001).

Supplementary Figure 8. A model depicting the functional competence of partially engaged complex.
A. Superimposition of β2AR-G protein crystal structure (PDB ID: 3SN6) with EM based structural model of β2V2R-βarr1 complex reveals an overlapping interface of the Gαs and the βarr1 on the receptor. β2AR is in grey, Gαs is in blue, Gβ is in yellow, Gγ is in pink and β-arrestin 1 is in orange. B. A schematic illustration to propose that the tail engaged complex is sufficient for receptor internalization, ERK binding and activation while fully engaged complex is required for receptor desensitization.