REMOVAL OP PARTIAL AOONISM PROM PARATHYROID HORMONE (PTH)-RELATED PROTEIN-(7-34)NH₂BY SUBSTITUTION OP PTH AMINO ACIDS AT POSITIONS 10 AND 11

Abstract

PTHrP(7-34)NH2 and [D-Trp12]PTHrP(7-34)NH2 have previously been shown to be shown to be more potent antagonists than the corresponding PTH peptide, [Tyr34]bPTH(7-34)NH2. However, these peptides also display partial agonism for adenylate cyclase activity in ROS 17/2.8 cells. In this study, design of a pure potent antagonist of PTH and PTHrP by removal of agonism from PTHrP(7-34)NH2 with retention of antagonist potency was accomplished. Since [Tyr34]bPTH(7-34)NH2 lacks agonist activity, we introduced two amino acids native to the PTH sequence into their respective positions in PTHrP and the potent D-Trp12 analog. [Asn10Leu11]- and [Asn10,leu11,D-Trp12]-PTHrP(7-34)NH2 were found to be 23- and 26-fold more potent as antagonists in ROS cells than PTHrP(7-34)NH2 and [D-Trp12]PTHrP(7-34)NH2, respectively. In addition, these peptides did not display partial agonism, even in an assay based on highly responsive cells pretreated with dexamethasone and pertussis toxin. In contrast, when the PTHrP sequence Asp10,Lys11 was inserted into [Tyr34]hPTH(7-34)NH2, antagonist potency declined by more than 6-fold and PTH-like agonist activity was installed. These results demonstrate that the activation domain of both PTH and PTHrP can be extended to include the 1-12 region and that the 10-12 region, in addition to the N-terminal hexapeptide, is important not only for receptor binding but also for hormonal signal transduction.

Nuclear targeting of a midregion PTHrP fragment is necessary for stimulating growth in breast cancer cells

“…Both PTHrP-(67-101) and PTHrP-(67-86) were found to significantly stimulate intracellular calcium ([Ca 21 ] i ) mobilisation in a dose dependent manner in both MCF-7 and MDA-MB231 cells (Figs. 4a and 4b respectively, To further demonstrate that the cellular responses seen with midregion PTHrP peptides were independent of PTHR1, MCF-7 cells were pre-incubated with a PTHR1 antagonist PTHrP-(7-34) 33 and [Ca 21 ] i mobilisation measured when stimulated by PTHrP-(67-101). We found PTHrP-(67-101) response not to be antagonised by PTHrP-(7-34) (Fig.…”

Parathyroid Hormone Rapidly Stimulates Hyaluronan Synthesis by Periosteal Osteoblasts in the Tibial Diaphysis of the Growing Rat

“…It contains 84 amino acids, the first 34 of which have most of its biological activity (2). Under normal physiological conditions PTH stimulates overall remodeling reactions in vivo by stimulating both bone formation and resorption while enhancing formation more (3).…”

“…Hyaluronan (HyA) 2 is a large polysaccharide consisting of several thousand repeats of a single disaccharide unit (1,4-glucuronic acid-␤-1,3-N-acetylglucosamine-␤) and can reach a relative molecular weight of several million daltons (11). It is an important component of several connective tissue matrices promoting tissue hydration and imparting high viscosity in certain tissue fluids (12), acting as a macromolecular scaffold for three-dimensional matrix assembly (13), modulating cellcell or cell-matrix interactions (14), affecting the differentiation of some progenitor cells (15,16), and regulating tissue inflammation via immune cell reactions (17).…”

Class II G Protein-Coupled Receptors and Their Ligands in Neuronal Function and Protection

“…The same effect is seen in other peptides, e.g., glucagon (Unson et al, 1993). This general scheme even applies to the distantly related PTH, which does not share a high primary sequence with GLP-1 at all, but nonetheless deletion of the first two amino acids also converts it into a potent competitive antagonist (Nutt et al, 1990). Interestingly, in this case, it has been postulated that the determinant responsible for selective signalling may not be equally located in the hormone.…”