Características que identifican al canal GAMA TV

From the data obtained from the alanine scan of ICL1, effects upon cAMP

production and iCa2+ mobilisation were observed (Figures 5.13-15, Tables

5.9-10), whilst observing minimal effects upon ERK1/2 activation (Figure 5.16, Table 5.11). Through quantifying these differences in terms of pathway bias, as performed in Chapters 3 and 4, it was possible to elucidate the differing effects that each residue has upon influencing differential G protein signalling. It was also observed that RAMP1-CLR heterodimers stimulated with CGRP display ~100-fold bias towards

preferentially signalling via Gαs and Gαq,over activating ERK1/2 (Figure

5.17). As each residue in ICL1 was mutated to alanine, it was possible to observe decreases in the extent of this bias, to the point that L169A and

C171A are equally biased towards iCa2+ mobilisation and ERK1/2

activation (Figure 5.17). Comparing the extent of bias between cAMP

production and iCa2+ mobilisation for each mutant uncovered an

interesting trend: Y165-K167A are all more biased towards iCa2+

mobilisation, over cAMP production (relative to ERK1/2 activation) (Figure 5.17). This then switches to being more biased towards cAMP production for S168-Q172A (Figure 5.17). An identical trend is observed when analysing relative bias, using ERK1/2 and WT CLR as references (Figure 5.18). This identified that relative to WT CLR, each of the mutants are more biased towards ERK1/2 activation over the other studied pathways

(Figure 5.18). However, Y165-K167A are still more biased to iCa2+

mobilisation over cAMP, with this switching for S168-Q172A (Figure 5.18). What is thus observed is that residues at the base of (Y165), and close to, TM1 (F166-K167) potentially play roles in the determination of

coupling to Gαs. This manifests itself as a bias towards mobilising iCa2+

when mutated to alanine, as these mutations have reduced the CLR’s ability to influence cAMP production. In contrast, the residues across the face of ICL1 (S168-C171) and at the base of TM2 (Q172) appear to play

cAMP production, due to the disruptive effects of mutation upon the

CLR’s ability to mobilise iCa2+. This work has therefore identified a

potential role of ICL1 in the determination of G protein specificity for the CLR, which may begin to explain how biased agonism occurs at this receptor.

Figure 5.17: Quantification of pathway bias (Δ (τ/Ka)) for WT and Y165- Q172A CLR.

Pathway bias (Δ (τ/Ka)) between cAMP production, iCa2+ mobilisation and ERK1/2 activation is shown for WT CLR, Y165A, F166A, K167A, S168A, L169A, S170A, C171A, Q172A, when co-expressed with RAMP 1 in HEK 293 cells, and stimulated with CGRP. 0.1! 1! 10! 100! 1000!WT Y165A F166A K167A S168A L169A S170A C171A Q172A cAMP! Ca2+! ERK1/2!

Δ (τ/Ka)

Figure 5.18: Relative pathway bias (ΔΔ (τ/Ka)) for WT and Y165-Q172A CLR.

Relative pathway bias (ΔΔ (τ/Ka)) between cAMP production, iCa 2+

mobilisation and ERK1/2 activation is shown for WT CLR, Y165A, F166A, K167A, S168A, L169A, S170A, C171A, Q172A, when co-expressed with RAMP 1 in HEK 293 cells, and stimulated with CGRP.

ΔΔ (τ/Ka)

5.11 Summary

This chapter has attempted to investigate the role of ICL1 in RAMP1-CLR activation and signalling. In order to achieve this, a saturation mutagenesis approach was utilised, mutating each residue (Y165-Q172), in turn, to: alanine, glutamic acid, glycine, histidine, isoleucine and arginine. The effect of these substitutions upon CGRP-mediated signalling, were characterised, when each mutant was co-expressed with RAMP1. To achieve this, classical pharmacological analyses: cAMP accumulation (for all substitutions), iCa2+ mobilisation and pERK1/2

assays (for alanine substitutions), were utilised, subsequently analysing their effects in terms of pathway bias.

As with previous studies (Bentrop et al, 1997, Wess et al, 1998), it was observed that mutating ICL1 has implications for cell surface expression of our receptor (Figure 5.7, Table 5.3). Interestingly, at some positions (Y165A, S168E, S168G, C171I) this actually results in an increase in expression, of between 10-50% over WT (Figure 5.7, Table 5.3). Some substitutions are well tolerated (F166A, S168A, S170A, F166E, F166H, S170H, Y165I, Y165R, K167R), with minimal impact upon expression (no greater than a 15% reduction) (Figure 5.7, Table 5.3). The most dramatic effects are observed for Q172H and L169R, with expressions of 10.05% and 4.35% that of WT, respectively (Figure 5.7, Table 5.3). Most other residues, except those specifically mentioned, tend to display levels of expression >30% that of WT (Figure 5.7, Table 5.3). Intriguingly, mutating L169 seems to be particularly susceptible to reducing expression, with all substitutions at this position resulting in reductions in expression of at least 66% (57.27% for isoleucine substitutions), relative to WT (Figure 5.7, Table 5.3). This residue is highly conserved amongst family B GPCRs (Figure 5.2); combined with the large reduction in cell surface expression we observe upon mutating this residue in the CLR, this suggests that a leucine at this position appears to play key roles in regulating expression. Whilst it is clear that reducing cell surface expression of each mutant will have effects upon signalling, it was still