The role of the cytosolic portion of the 7th transmembrane helix of calcitonin receptor-like receptor (CLR) in coupling to Gs

Calcitonin receptor-like receptor (CLR) is a G-protein coupled receptor (CGRP) which interacts with RAMP1 to generate the CGRP receptor (CGRPR) and RAMP2 to give the adrenomedullin (AM)-1 receptor (AM1R) [1]. We have previously explored the role of individual residues throughout the entire transmembrane (TM) portion of the receptor [2]. In this study, we present an alanine scan of the cytosolic half of the 7th TM helix (TM7), investigating how this influences the ability of the receptor to couple to Gs by producing cAMP.

Each residue in HA-tagged human (h) CLR from V380 to E390 was replaced by alanine by site-directed mutagenesis [2]. The receptors were transiently transfected into Cos 7 cells with either RAMP1 or RAMP2 [2] and concentration response curves were constructed to either h alpha CGRP or h AM (10-11M to 10-6M), measuring cAMP by Alphascreen [3]. Concentration-response curves were fitted to give Emax, minimum and pEC50. Cell surface expression was measured by ELISA [2]. Statistical analysis was by one-way ANOVA followed by Dunnett’s test.

All mutants expressed at levels greater than 70% compared to wild type (WT) in the presence of RAMP1 or RAMP2, apart from N388A-CLR/RAMP1, which showed 62 ± 1% expression (n=3). For CGRP on the CGRP receptor, the only significant reduction in pEC50 was also for N388A (9.55 ± 0.06 v 10.55 ± 0.05, n=3, P<0.05). There were no changes in Emax. There were no significant changes in either Emax or pEC¬50 for AM at the AM1R. For AM at the CGRP receptor, the largest change was a reduction in Emax with E390A, which was 71 ± 1% of WT (n=3); there were reductions in the pEC50 for this and also F384A and N388A (WT 8.93±0.14, E390A 7.50±0.05*, N388A 7.74±0.09*, F384A 7.91±0.04*, n=3).

These data suggest that individual residues in the cytosolic half of TM7 of CLR play only minor roles in coupling to Gs, at least as far as can be assessed by alanine mutagenesis; indeed N388 and E390 may be better considered to be at the junction with helix 8. It is possible that collectively they are more important. The results are of some interest as it has been claimed that F387, normally conserved as a tyrosine in other family B GPCRs, functions as part of a conserved network in Gs coupling [4]. Under the conditions of our experiments, we were unable to confirm this. This suggests that the role of this particular switch depends on the receptor under examination.

This work was supported by BBSRC grants BB/M006883 and BB/M007529.

References

1. Poyner, DR et al (2002). Pharmacol Rev 54: 233-246.

2. Woolley, MJ et al (2013). J R Soc Interface. 10: 20130589

3. Watkins, HA et al (2014) Br J Pharmacol. 171: 772-88.

4. Wootten, D et al (2013) Proc Natl Acad Sci U S A. 110: 5211-6.