Autoactivation of type-1 parathyroid hormone receptors containing a tethered ligand

Interactions between the N-terminal residues of parathyroid hormone (PTH) a nd the region of the PTH receptor containing the extracellular loops and tr ansmembrane domains are thought to be critical for receptor activation. We evaluated this hypothesis by replacing the large N-terminal extracellular d omain of the human type 1 PTH receptor (hP1Rc-WT) with residues 1-9 of PTH (AVSEIQLMH) using a tetraglycine linker between His-9 of the ligand and Glu -182 of the receptor near the extracellular terminus of transmembrane domai n-1. Expression of this construct, hP1Rc-Tether(1-9), in COS-7 cells result ed in basal cAMP levels that were 10-fold higher than those seen in control cells transfected with hP1Rc-WT, Extending the ligand sequence to include Asn-10 and the activity-enhancing substitution of Leu-11 --> Arg yielded hP 1Rc-[Arg(11)]Tether(1-11), for which we observed basal cAMP levels that wer e 50-fold higher than those seen with P1Rc-WT, An alanine-scan analysis of hP1Rc-[Arg(11)]Tether(1-11) revealed that Gln-6 and His-9 were not critical for autoactivation, whereas Val-2, Ile-5, and Met-8 were. The data show th at tethered PTH/PTH receptors can autoactivate. Analysis of the structure-a ctivity relationships in these tethered receptor constructs can provide new information concerning how the N-terminal residues of PTH interact with th e extracellular loops and transmembrane regions of the PTH-1 receptor, part icularly in regard to receptor activation.