Biological activity in vitro and in vivo of peptides corresponding to the third intracellular loop of thyrotropin receptor

Abstract

In recent years, one of the rapidly developing directions in molecular endocrinology and nanobio technology is the peptide strategy, which has become widely used for studying the molecular mechanisms of transduction of hormonal signals into the cell and for designing highly selective and highly effective reg ulators of hormonal signaling systems [1,2]. It is based on the search and development of peptides cor responding in structure to the functionally important parts of signaling proteins-receptors, heterotrimeric G proteins, and enzymes that generate second mes sengers. The most promising direction in the peptide strategy is the synthesis and study of peptides derived from serpentine type receptors, because at the level of these receptors not only the specific recognition of an external signal but also the choice of the pathway of its transduction into the cell takes place and, therefore, the intracellular targets of the hormone are deter mined. We and other authors demonstrated that pep tides corresponding to the cytoplasmic loops (CLs) of serpentine type receptors selectively interact with G proteins, trigger signaling cascades in the absence of hormone, affect the transduction of the signal gener ated by them through a homologous receptor, thus functioning as regulators of intracellular signaling [3][4][5][6][7][8][9].One of the urgent problems of modern endocrinol ogy is the search for an effective regulator of the thy roid gland and the entire hypothalamic pituitary thy roid axis, which act at the stage of activation of the thyroid stimulating hormone (TSH) receptor by the hormone. Disturbances that occur at this signal trans duction stage lead to a wide spectrum of thyroid gland diseases, including autoimmune thyroiditis and thy roid cancer. The purpose of the study was to develop selective regulators of the TSH sensitive signaling pathways on the basis of the peptides derived from the C terminal region 612-627 of the third intracellular loop (C ICL 3) of the TSH receptor. We also studied the activity of the peptides in vitro by their effects on the basal and hormone stimulated level of GTP bind ing of heterotrimeric G s and G q proteins, which are components of these cascades, and in vivo by the effect of intranasal administration of peptides on the level of thyroid hormones in the blood plasma of experimental animals. It should be noted that mutations in the C ICL 3 of the TSH receptor disturb its interaction with G s proteins and lead to loss of the ability of the mutant receptor to stimulate adenylate cyclase activity and cAMP dependent signaling pathways [10].Using solid phase peptide synthesis, we synthe sized the peptide Gln Tyr Asn Pro Arg Asp Lys Asp Thr Lys Ile Ala Lys Arg Nle Ala 612-627 Lys Ala amide (I), in which Met 626 is replaced with norleu cine having similar physicochemical properties. We also synthesized a palmitoylated analogue of this peptide (II), in which the side ε amino group of the lysine residue following Ala 627 is modified with the palmitic acid residue. The peptides were syn...

Allosteric Regulation of G-Protein-Coupled Receptors: From Diversity of Molecular Mechanisms to Multiple Allosteric Sites and Their Ligands

“…In addition to allosteric sites localized in the ECLs and TMD, such sites are also located in the ICLs of TSHR, as evidenced by our results on the stimulating effect on TSHR activity of pepducin, which corresponds to region 612-627 of TSHR ICL3 [353,354] (Figure 3). Pepducin 612-627(Palm) modified with palmitate at the C-terminus stimulated the AC activity in rat thyroid membranes, and its action was specific for TSHR and was not detected in membranes where TSHR was absent [353]. When administered intranasally to rats, 612-627(Palm) increased the level of thyroid hormones in them, and this effect was dose-dependent [354].…”

“…When administered intranasally to rats, 612-627(Palm) increased the level of thyroid hormones in them, and this effect was dose-dependent [354]. The obtained data suggest that 612-627(Palm) is an intracellular allosteric TSHR agonist, and hydrophobic palmitate is critical for its activity, providing the transport of pepducin into the cell, since the non-palmitoylated analog was inactive [353,354].…”

Thyroid-Stimulating Hormone Receptor: the Role in the Development of Thyroid Pathology and Its Correction

“…Along with allosteric regulators interacting with the transmembrane allosteric site, it seems prom ising to develop ligands for allosteric sites local ized in the TSHR cytoplasmic loops, primarily in its second and third cytoplasmic loops, which contain key molecular determinants responsible for the interaction with G proteins. In this regard, palmitoylated peptide fragments of the TSHR third cytoplasmic loop are promising, as they are able to stimulate the activity of the receptor in the absence of TSH by changing the conformation of its cytoplasmic allosteric sites and thereby to modulate the interaction of the receptor with G proteins [67][68][69].…”

GPCR-Peptides: Prospective Use in Biology and Medicine

“…Many data are available now on ICL-peptides as regulators and modulators of functioning of endocrine system in the in vitro and in vivo conditions [19][20][21][22][34][35][36][37][38]. C-palmitoylated pepducin 612-627-K(Pal)A corresponding to C-terminal part of the third ICL of thyroid-stimulating hormone (TSH) receptor in vitro stimulated the basal activity of adenylyl cyclase and GTP-binding capacity of G sproteins, the components of TSH-sensitive adenylyl cyclase signaling system, and reduced the stimulating effects of TSH on this system in the thyroidal membranes [22].…”

“…C-palmitoylated pepducin 612-627-K(Pal)A corresponding to C-terminal part of the third ICL of thyroid-stimulating hormone (TSH) receptor in vitro stimulated the basal activity of adenylyl cyclase and GTP-binding capacity of G sproteins, the components of TSH-sensitive adenylyl cyclase signaling system, and reduced the stimulating effects of TSH on this system in the thyroidal membranes [22]. The 5-days treatment of rats with intranasally administered 612-627-K(Pal)A resulted in a significant and sustained increase of free T 4 level and a decrease of TSH level [37]. These data furnish evidence that TSH receptor-derived pepducins can be used to develop new regulators of the thyroid functions.…”