Investigating pharmacological interactions between mGlu1 and mGlu5 using a constitutive heterodimer construct

Following the discovery that metabotropic glutamate receptor 5 (mGlu5) exists as a covalently bonded homodimer(1), in house data lead us to believe that mGlu5 may form a heterodimer with mGlu1. Using mGlu1 and mGlu5 constructs with C terminals modified with GABAB tails, we hoped to understand whether these receptors would interact pharmacologically as a dimer.

The pharmacological properties of mGlu5 specific positive allosteric modulators, LSN-2814617 and CDPPB, were determined for mGlu1, mGlu5, mGlu5 constitutive homodimer (mGlu5.5), and mGlu1/5 constitutive heterodimer (mGlu1.5) cell lines using a Fluorometric Imaging Plate Reader (FLIPR). Briefly, stable cell lines expressing the above constructs were plated onto poly-D-lysine coated 96 well plates at 50,000 cells/well and incubated for 24 hours at 37 ºC with 95 % air and 5 % CO2. The cells were then incubated in HBSS supplemented with 10 mM HEPES and 10 μM Fluo-4 AM and incubated for 1 hour at room temperature in the dark. A range of concentrations of group I selective agonist (S)-3,5-DHPG (150 nM to 30 μM) were assayed in combination with a range of concentrations of either LSN-2814617 (10 nM to 10 μM) or CDPPB (3 nM to 3 μM). Intracellular calcium levels were monitored before and after the addition of compounds, with data collection ranging from 1 image every second to 1 image every 3 seconds. Further assays were performed as above using an EC90 concentration of (S)-3,5-DHPG (10 μM) to assess a range of concentrations of the highly selective mGlu5 negative allosteric modulator MPEP (3 pM to 10 μM). Data was analysed using the operational ternary complex model with Graphpad Prism v6.0.

Our data showed that the mGlu5 and mGlu5.5 constructs had insignificant differences in their pharmacological properties with respect to LSN-2814617 (αβ = 3.95 ± 0.32 for mGlu5, 3.99 ± 0.39 for mGlu5.5; p > 0.05 two-tailed Student’s t-test), however, mGlu1.5 was significantly different to the wild-type (αβ = 1.68 ± 0.37; p = 0.009 two-tailed Student’s t-test versus mGlu5). The properties of CDPPB were not significantly different between mGlu5 and mGlu5.5 (αβ = 8.84 ± 0.38 for mGlu5, 13.9 ± 1.13 for mGlu5.5; p > 0.05 two-tailed Student’s t-test), but again mGlu1.5 was significantly different to the wild-type (αβ = 3.45 ± 1.26; p = 0.005 two-tailed Student’s t-test versus mGlu5). Similarly, there was no significant difference in the ability of MPEP to inhibit at (S)-3,5-DHPG response at the mGlu5 and mGlu5.5 constructs (EC50 = 8.89 ± 0.05 for mGlu5, 8.59 ± 0.02 for mGlu5.5; p > 0.05 two-tailed Student’s t-test). There was a significant difference in the ability of MPEP to inhibit an (S)-3,5-DHPG response at the mGlu1.5 compared to the wild-type and mGlu5.5 (EC50 = 7.86 ± 0.09; p = <0.005). MPEP only inhibited 50% of the (S)-3,5-DHPG response in the mGlu1.5 assays, but acted as a full inhibitor at the wild-type and mGlu5.5 homodimer.

Our experiments have shown that there are significant differences between the pharmacological profile of allosteric modulators acting at the mGlu5 homodimer compared to a mGlu1.5 heterodimer. The mGlu1 part of the heterodimer appears to reduce the activity of allosteric modulators at the mGlu5 receptor. This is unlikely to be due to the modification of the C-terminal tail, as this effect was not present in the mGlu5.5 homodimer construct. Further experiments, including assessing the binding properties of these constructs could elucidate whether these effects are by binding-site inhibition or functional inhibition, and whether the dimer exists in vivo.

(1) Romano et al (1998). J Biol Chem 271(45): 28612–28616.