Extracellular signal-regulated kinase activation by parathyroid hormone in distal tubule cells

The PTH receptor ( PTH1R) activates multiple signaling pathways, including extracellular signal- regulated kinases 1 and 2 ( ERK1/ 2). The role of epidermal growth factor receptor ( EGFR) transactivation in ERK1/ 2 activation by PTH in distal kidney cells, a primary site of PTH action, was characterized. ERK1/ 2 phosphorylation was stimulated by PTH and blocked by the EGFR inhibitor, AG1478. Upon PTH stimulation, metalloprotease cleavage of membrane- bound heparinbinding fragment ( HB- EGF) induced EGFR transactivation of ERK. Conditioned media from PTH- treated distal kidney cells activated ERK in HEK- 293 cells. AG1478 added to HEK- 293 cells ablated transactivation by conditioned media. HB- EGF directly activated ERK1/ 2 in HEK- 293 cells. Pretreatment of distal kidney cells with the metalloprotease inhibitor GM- 6001 abolished transactivation of ERK1/ 2 by PTH. The role of the PTH1R COOH terminus in PTX- sensitive ERK1/ 2 activation was characterized in HEK- 293 cells transfected with wild- type PTH1R, with a PTH1R mutated at its COOH terminus, or with PTH1R truncated at position 480. PTH stimulated ERK by wild- type, mutated and truncated PTH1Rs 21-, 27and 57- fold, respectively. Thus, the PTH1R COOH terminus exerts an inhibitory effect on ERK activation. EBP50, a scaffolding protein that binds to the PDZ recognition domain of the PTH1R, impaired PTH but not isoproterenol or calcitonin- induced ERK activation. Pertussis toxin inhibited PTH- stimulated ERK1/ 2 by mutated and truncated PTH1Rs and abolished ERK1/ 2 activation by wild- type PTH1R. We conclude that ERK phosphorylation in distal kidney cells by PTH requires PTH1R activation of G(i), which leads to stimulation of metalloprotease- mediated cleavage of HB- EGF and transactivation of the EGFR and is regulated by EBP50.