MUTATIONS IN THE 2ND CYTOPLASMIC LOOP OF THE RAT PARATHYROID-HORMONE (PTH) PTH-RELATED PROTEIN-RECEPTOR RESULT IN SELECTIVE LOSS OF PTH-STIMULATED PHOSPHOLIPASE-C ACTIVITY/

To define the structural requirements of the parathyroid hormone (PTH) /PTH-related protein (PTHrP) receptor necessary for activation of phos pholipase C (PLC), receptors with random mutations in their second cyt oplasmic loop were synthesized, and their properties were assessed. A mutant in which the wild type (WT) rat PTH/PTHrP receptor sequence EKK Y (amino acids 317-320) was replaced with DSEL had little or no PTH-st imulated PLC activity when expressed transiently in COS-7 cells, but i t retained full capacity to bind ligand and to generate cAMP. This phe notype was confirmed in LLC-PK1 cells stably expressing the DSEL mutan t receptor, where both PTH-stimulated PLC activity and sodium-dependen t phosphate co-transport were essentially abolished. Individual mutati ons of these four residues point to a critical role for Lys-319 in rec eptor-G protein coupling. PTH-generated IPs were reduced to 27 +/- 13% when K319E, compared with the WT receptor, and PLC activation was ful ly recovered in a receptor revertant in which Glu-319 in the DSEL muta nt cassette was restored to the WT residue, Lys. Moreover, the WT rece ptor and a mutant receptor in which K319R had indistinguishable proper ties, thus suggesting that a basic amino acid at this position may be important for PLC activation. All of these receptors had unimpaired ca pacity to bind ligand and to generate cAMP. To ensure adequacy of G al pha(p)-subunits for transducing the receptor signal, G alpha(p) was ex pressed in HER293 and in LLC-PK1 cells together with either WT recepto rs or receptors with the DSEL mutant cassette. PTH generated no inosit ol phosphates (LPs) in either HEK293 or LLC-PK1 cells, when they expre ssed DSEL mutant receptors together with G alpha(p). In contrast, PTH generated 2- and 2.5-fold increases in IPs, respectively, when these c ells co expressed both the WT receptor and GLYB Thus, generation of IP s by the activated PTR/PTHrP receptor can be selectively abolished wit hout affecting its capacity to generate cAMP, and Lys-319 in the secon d intracellular loop is critical for activating the PLC pathway, Moreo ver, alpha-subunits of the G(q), family, rather than py-subunits, tran sduce the signal from the activated receptor to PLC, and the PLC, rath er than the adenylyl cyclase, pathway mediates sodium-dependent phosph ate cotransport in LLC-PK1 cells.