Figure 2. Functional test of receptor phosphorylation.
The “Extra Meta II” assay measures the stabilisation of Meta II (λmax: 380 nm) over its precursor Meta I (λmax: 480 nm) by absorbance spectroscopy. In the absence of any binding partner, very little Meta II evolved (black trace). In the presence of the high-affinity analogue peptide derived from the C-terminus of the α-subunit of transducin (Gtα-peptide, VLEDLKSCGLF, 350 μM), 100% of receptors were stabilised as Meta II (blue trace). In the presence of arrestin (10 μM), all receptors were also stabilised as Meta II (red trace). Hence, all receptors in our preparation were sufficiently phosphorylated to bind arrestin as Meta II-P. A high level of receptor phosphorylation is indicated by the fast rate of arrestin binding, which is comparable to Gtα-peptide. This assay has previously been established as an indicator of functional receptor phosphorylation for studies of arrestin binding22.