Intracellular Loop 2 Peptides of the Human 5HT1a Receptor are Differential Activators of Gi

Abstract

Peptide mimics of intracellular loop 2 (ic2) of the human 5HT1a receptor have been studied with respect to their ability to inhibit agonist binding via interference with receptor-G-protein coupling. These peptides give shallow concentration-effect relationships. Additionally, these peptides have been studied with respect to their ability to trigger the signal transduction system of this Gi-coupled receptor. Two signaling parameters have been quantified: concentration of intracellular cAMP and changes in incorporation into the G protein of a stable analog of GTP. In both cases, peptide mimics near midloop of ic2 actually show agonist activity with efficacy falling off toward both loop termini near TM 3 and TM 4. Previous results have suggested that the loop region near the TM3/ic2 interface is primarily responsible for receptor-G-protein coupling, while the current result emphasizes the mid-ic2 loop region's ability to activate the G protein following initial coupling. A limited number of peptides from the receptor's TM5/ic3 loop vicinity were also studied regarding agonist inhibition and G-protein activation. These peptides provide additional evidence that the human 5HT1a receptor, TM5/ic3 loop region, is involved in both coupling and activation actions. Overall, these results provide further information about potential pharmacological intervention and drug development with respect to the human 5HT1a receptor/G-protein system. Finally, the structural evidence generated here provides testable models pending crystallization and X-ray analysis of the receptor.

Figure 1

P21 noncentration-dependent displacement of bound 8-OH-DPAT. This curve represents the change in specific binding of [³H]-8-OH-DPAT, a 5HT1aR agonist, to the receptor in the presence of various concentrations of the ic2 peptide mimic P21. Nominal binding of agonist at control levels was 400 fmoles/mg protein.

Figure 2

ic2 peptide effect on γ-[³⁵S]-GTP incorporated into Gi, a measure of G-protein activation. Control is the basal amount of γ-[³⁵S]-GTP incorporated into Gi in CHO cells expressing the human 5HT1aR, set as 100%. The Y-axis is the percent of specifically bound (total minus nonspecific) γ-[³⁵S]-GTP. All other treatments are percents of the control value. All peptides are 30 uM concentration and 5HT 10⁻⁷ M concentration. Error is expressed as SEM.

Figure 3

ic2 peptide effect on forskolin-stimulated cAMP production, a measure of activated-G-protein regulation of adenylyl cyclase. Forskolin (FSK) stimulated cAMP production by adenylyl cyclase (AC) is in CHO cells expressing the human 5HT1aR. FSK (30 uM) is the control, which is set to 100%. All other treatments are expressed as a percent of the control value. Peptide concentrations are 30 uM. All treatments include isobutylmethylxanthine (IBMX) an inhibitor of the metabolism of cAMP by phosphodiesterase. Error is expressed as SEM.

Figure 4

P12 and P13-stimulated incorporation of γ-[³⁵S]-GTP control is the basal amount of γ-[³⁵S]-GTP incorporated into Gi in CHO cells expressing the human 5HT1aR set as 100%. The Y axis is the percent of specifically bound γ-[³⁵S]-GTP. All other treatments are percents of the control value. Peptide concentrations are 30 uM. *P < 0.01 P12 versus control; ^€ P < 0.01 5HT versus 5HT/P12. *P13 versus control P < 0.01; ^€5HT versus 5HT/P13 P < 0.01.

Figure 5

P12 and P13 effect of forskolin-stimulated cAMP production forskolin (FSK) stimulated cAMP production by adenylyl cyclase (AC) in CHO cells expressing the human 5HT1aR. These experiments were a measure of second messenger regulation by G protein. FSK is the control, which is set to 100%. All other treatments are expressed as a percent of the control value. Peptide concentrations are 30 uM. All treatments include isobutylmethyl xanthine (IBMX), an inhibitor of the metabolism of cAMP. 5HT versus 5HT/P12 and 5HT versus 5HT/P13 *P < 0.05.