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FRET ͉ G protein-coupled receptor ͉ time-resolved signaling ͉ real-time binding ͉ ligand-receptor interaction P arathyroid hormone (PTH) is an 84-aa peptide that regulates concentrations of calcium and phosphate in blood and is of paramount importance in renal and bone metabolism (1). PTH is secreted from the parathyroid glands in response to hypocalcemia and elicits its physiological effects in kidney and bone through the activation of a G protein-coupled receptor, the PTH͞PTH-related receptor (PTHR) (2). In response to PTH this receptor leads to activation of adenylyl cyclases (via G s protein) that in turn increase the intracellular second messenger cAMP, which activates the protein kinase A (PKA)-signaling pathway. PTHR also activates the phospholipase C͞protein kinase C-signaling pathways (via G q protein), although less efficiently than the PKA-signaling pathway (3). PTH(1-34), a synthetic PTH fragment with the first 34 aa of PTH that holds the same pharmacological properties in regard to the regulation of calcium homeostasis as the full-length hormone (4), has recently become a drug (the generic name is teriparatide) approved by the U.S. Food and Drug Administration to treat osteoporosis (5, 6). The resolution of the mode of interaction of PTH(1-34) to its receptor is critical for understanding the mechanism of action of the hormone and for further progressing in drug development to treat osteoporosis.We recently showed that activation of PTHR by binding of PTH(1-34) proceeds by fast conformational changes (time constant Ϸ 1 s) as the receptor switches from a resting to an active state (7). To further our understanding on the PTHR interaction process we resolved the temporal events of PTH(1-34) binding to PTHR in living cells. We measured kinetics of association by FRET between tetramethyl-rhodamine (TMR) (the acceptor fluorophore) conjugated to PTH(1-34) and GFP (the donor fluorophore) inserted into the N-terminal domain of the receptor (Fig. 1A). This study reports the analysis of these kinetics and reveals a two-step process for PTH(1-34) binding, where the first phase mirrored agonist binding to the receptor N-domain with a fast association rate and the second phase reflected association to the J-domain, exhibiting much slower kinetics but temporally coupled to receptor activation. Materials and MethodsPeptides. Human PTH(1-34) was purchased from Bachem, and the radioligand [ 125

Advanced glycation endproducts (AGEs), which accumulate on long-lived proteins and protein deposits (amyloids), induce the expression of proinflammatory cytokines through NF-U UB-dependent pathways. Hyaluronic acid with a molecular weight above 1.2 MDa (HMW-HA) inhibits the AGEinduced activation of the transcription factor NF-U UB and the NF-U UB-regulated cytokines interleukin-1K K, interleukin-6 and tumor necrosis factor-K K. Since the molecular weight of hyaluronic acid in humans decreases with age and under conditions of oxidative stress, it is likely that the protective effect of HMW-HA against AGE-induced cellular activation is lost at sites of chronic inflammation and in older age.z 1999 Federation of European Biochemical Societies.

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