Mutations in the second 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 phospholipase C (PLC), receptors with random mutations in their second cytoplasmic loop were synthesized, and their properties were assessed. A mutant in which the wild type (WT) rat PTH/PTHrP receptor sequence EKKY (amine acids 317320) was replaced with DSEL had little or no PTH-stimulated PLC activity when expressed transiently in COS-7 cells, but it retained full capacity to bind ligand and to generate cAMP. This phenotype was confirmed in LLC-PK1 cells stably expressing the DSEL mutant receptor, where both PTH- stimulated PLC activity and sodium-dependent phosphate co-transport were essentially abolished. Individual mutations of these four residues point to a critical role for Lys-319 in receptor-G protein coupling. PTH-generated IPs were reduced to 27 ± 13% when K319E, compared with the WT receptor, and PLC activation was fully recovered in a receptor revertant in which Glu-319 in the DSEL mutant cassette was restored to the WT residue, Lys. Moreover, the WT receptor and a mutant receptor in which K319R had indistinguishable properties, thus suggesting that a basic amine acid at this position may be important for PLC activation. All of these receptors had unimpaired capacity to bind ligand and to generate cAMP. To ensure adequacy of Gα(q)-subunits for transducing the receptor signal, Gα(q) was expressed in HEK293 and in LLC-PK1 cells together with either WT receptors or receptors with the DSEL mutant cassette. PTH generated no inositol phosphates (IPs) in either HEK293 or LLC-PK1 cells, when they expressed DSEL mutant receptors together with Gα(q). In contrast, PTH generated 2- and 2.5-fold increases in IPs, respectively, when these cells co-expressed both the WT receptor and Gα(q). Thus, generation of IPs by the activated PTHrP receptor can be selectively abolished without affecting its capacity to generate cAMP, and Lys-319 in the second intracellular loop is critical for activating the PLC pathway. Moreover, a-subunits of the G(q) family, rather than βγ-subunits, transduce the signal from the activated receptor to PLC, and the PLC, rather than the adenylyl cyclase, pathway mediates sodium-dependent phosphate cotransport in LLC-PK1 cells.