Low Affinity Analogs of Thyrotropin-releasing Hormone Are Super-agonists

Abstract

We show that several analogs of thyrotropin-releasing hormone (TRH) are more efficacious agonists at TRH receptors R1 and R2 than TRH itself. The apparent efficacies of the analogs were inversely related to their potencies and were independent of the nature of the modifications in TRH structure. In studies in intact cells, we showed that the differences in apparent efficacies were not due to differences in G-protein coupling, receptor desensitization, or recycling. Moreover, the differences in efficacies persisted in experiments using accessory protein-free membranes. We conclude that the efficacy differences of TRH analogs originated from the enhanced ability of TRH-R complexed to the low affinity agonists to directly activate G-protein(s), and not by a modulation of the activity of accessory proteins, and propose possible mechanisms for this phenomenon.Thyrotropin-releasing hormone (TRH) 2 receptors (TRH-Rs) are members of the rhodopsin-like family (family A) of G-protein coupled receptors. TRH-Rs couple primarily to the G q/11 subfamily of G-proteins and mediate the intracellular release of Ca 2ϩ through the activation of the inositol phosphate (IP) pathway (1). There are two subtypes of TRH-Rs, TRH-R1 and TRH-R2, that share about 50% sequence homology (2, 3). The physiological significance of the existence of two kinds of TRH-Rs remains unknown. TRH-R1 and TRH-R2 exhibit subtle functional differences varying in the level of stimulated and basal signaling, the rate of internalization, and the ability to couple to G-proteins other than G q/11 (2-4). The ligand binding affinities of TRH analogs to the two receptors, however, are very similar (2). TRH is the natural agonist for both TRH-Rs, and numerous synthetic analogs of TRH have been shown to stimulate both receptors (2). Except for substitution of His by 1-methyl-His, all substitutions within TRH result in analogs with reduced affinities, but all are agonists.The nature of the molecular changes that are responsible for TRH-R activation remain mainly unknown. The absence of a working hypothesis for the mechanism of TRH-R activation precludes the use of a rational approach to develop new agonists for these receptors. Therefore, study of structure-activity relationships of the known TRH-R1/R2 agonists may contribute to a better understanding of the structural basis of the efficacies of TRH-R-agonist complexes, which is necessary for development of more efficient (specific) modulators of TRH-R activity. In this work, we demonstrate a unique pharmacological profile of a series of TRH analogs in which affinities (potencies) of the compounds are related in an inverse mode to their ability to activate TRH-R1/R2. A corollary of these observations is that certain TRH analogs act as "superagonists" for TRH-R1/R2. We propose possible mechanisms for this effect.

Thyrotropin Receptor Stimulates Internalization-Independent Persistent Phosphoinositide Signaling

“…The generation of a HEK-EM293 cell lines stably expressing human TSHR (HEK-TSHR cells) or human TRH receptor were described previously (Engel et al, 2006;Neumann et al, 2009). Cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 10 g/ml streptomycin and 250 g/ml hygromycin B (both from Invitrogen, Carlsbad, CA) at 37°C in a humidified 5% CO 2 incubator.…”

Long Receptor Residence Time of C26 Contributes to Super Agonist Activity at the Human β2 Adrenoceptor

“…shares properties with other synthetic ligands that have been shown to have higher intrinsic activity than their respective endogenous ligands, such as the non-peptide growth hormone secretagogue MK-677 (Bennett et al, 2009;Holst et al, 2005) and the thyrotropin-releasing hormone analogue R-Des-TRH (Engel et al, 2006).…”

“…If such a case were true, the maximal asymptote of the concentration-response curves of endogenous agonists, fitted to experimental data (Strange, 2008) (intrinsic activity, E max ), would always be equal to the system maximum. However, there are a class of synthetic ligands such as MK-677 at the GNRH receptor or R-Des-TRH at the TRH receptor (Bennett et al, 2009;Engel et al, 2006) that have been demonstrated to produce higher maximal responses than their respective endogenous ligands, leading to the term super agonist being coined. Such terminology has been met with mixed views in the pharmacology community, and the unique pharmacology of super agonists is now the subject of increasing interest and debate.…”

Synthesis of CNS active thyrotropin-releasing hormone (TRH)-like peptides: Biological evaluation and effect on cognitive impairment induced by cerebral ischemia in mice

“…12,24 Jain et al have reported the synthesis of TRH analogs in which both pGlu and central histidine residue was simultaneously modified. 25,26 Gershengorn and Jain et al 27,28 have showed for the first time that certain TRH analogs were more efficacious agonists at TRH-R1 and TRH-R2 than TRH itself. They found that decrease in the affinities caused by TRH analogs correlate inversely with the analog ability to activate TRH-Rs.…”