RNA-editing of the 5-HT(2C) receptor alters agonist-receptor-effector coupling specificity

Abstract

1. The serotonin(2C) (5-HT(2C)) receptor couples to both phospholipase C (PLC)-inositol phosphate (IP) and phospholipase A(2) (PLA(2))-arachidonic acid (AA) signalling cascades. Agonists can differentially activate these effectors (i.e. agonist-directed trafficking of receptor stimulus) perhaps due to agonist-specific receptor conformations which differentially couple to/activate transducer molecules (e.g. G proteins). Since editing of RNA transcripts of the human 5-HT(2C) receptor leads to substitution of amino acids at positions 156, 158 and 160 of the putative second intracellular loop, a region important for G protein coupling, we examined the capacity of agonists to activate both the PLC-IP and PLA(2)-AA pathways in CHO cells stably expressing two major, fully RNA-edited isoforms (5-HT(2C-VSV), 5-HT(2C-VGV)) of the h5-HT(2C) receptor. 2. 5-HT increased AA release and IP accumulation in both 5-HT(2C-VSV) and 5-HT(2C-VGV) expressing cells. As expected, the potency of 5-HT for both RNA-edited isoforms for both responses was 10 fold lower relative to that of the non-edited receptor (5-HT(2C-INI)) when receptors were expressed at similar levels. 3. Consistent with our previous report, the efficacy order of two 5-HT receptor agonists (TFMPP and bufotenin) was reversed for AA release and IP accumulation at the non-edited receptor thus demonstrating agonist trafficking of receptor stimulus. However, with the RNA-edited receptor isoforms there was no difference in the relative efficacies of TFMPP or bufotenin for AA release and IP accumulation suggesting that the capacity for 5-HT(2C) agonists to traffic receptor stimulus is lost as a result of RNA editing. 4. These results suggest an important role for the second intracellular loop in transmitting agonist-specific information to signalling molecules.

Figure 1

Comparison of concentration response curves to 5-HT between non-edited (5-HT_2C-INI) and RNA-edited (5-HT_2C-VSV and 5-HT_2C-VGV) 5-HT_2C receptor isoforms. CHO cells expressing stably the 5-HT_2C-INI, 5-HT_2C-VSV or 5-HT_2C-VGV receptor isoforms were incubated with various concentrations of 5-HT for 10 min followed by measurement of IP accumulation or AA release simultaneously from the same multiwell. Data shown are normalized to the per cent of E_max from each individual experiment and represent the mean of 4 – 5 experiments measured in triplicate. Individual concentration curves were fit to the logistic equation 2 described in Methods to determine E_max and EC₅₀ values (provided in Table 1).

Figure 2

Relative efficacy of LSD for AA release and IP accumulation in cells expressing the 5-HT_2C-VSV isoform at high densities. Cells were incubated with 300 nM LSD (maximal concentration) for 10 min followed by simultaneous measurement of AA release or IP accumulation. Bars show the intrinsic activity of LSD, referenced to the maximal 5-HT response determined in the same experiment, and represent the mean±standard error of three experiments. 5-HT-mediated AA release was 70±7% and 85±7% and 5-HT-mediated IP accumulation was 293±33% and 429±40%, for cells expressing 2300 and 7600 fmol mg⁻¹, respectively.

Figure 3

Comparison of relative efficacies of bufotenin, TFMPP and LSD between 5-HT_2C-INI and 5-HT_2C-VGV receptor isoforms. Cells were incubated for 10 min with maximal concentrations of LSD (300 nM), bufotenin (10 μM for 5-HT_2C-INI and 30 μM for 5-HT_2C-VGV), TFMPP (10 μM for 5-HT_2C-INI and 30 μM for 5-HT_2C-VGV) or 5-HT (1 μM for 5-HT_2C-INI and 3 μM for 5-HT_2C-VGV). AA release and IP accumulation were measured simultaneously from the same multiwell. Data were normalized to the 5-HT response determined in the same experiment and represent the mean±s.e.mean of 3 – 6 experiments. 5-HT-mediated responses for the 5-HT_2C-INI isoform were 85±13% and 285±47%, for the 5-HT_2C-VGV clone #1, 86±15% and 169±10% and for 5-HT_2C-VGV clone #2, 47±6% and 189±4%, AA release and IP accumulation, respectively. ^*Denotes statistical significance between agonist-mediated AA release and IP accumulation.