Detection of heterologous Gs-coupled receptor activity in LLC-PK1 cells based on expression of urokinase-type plasminogen activator.

Abstract

In renal epithelial cells LLC-PK1 () expression of the urokinase-type plasminogen activator (uPA) gene is strongly induced by first messengers that stimulate intracellular CAMP synthesis (). The increase of CAMP leads to a 200-fold activation of uPA gene transcription (). In response to optimal concentrations of the peptide hormone calcitonin, i.e., for which the cells express a Gs-coupled receptor (,), high levels of uPA protein are synthesized and secreted. The uPA protein is a serine/threonine protease that converts the zymogen plasminogen into plasmin, a protease with a wide substrate specificity (). The uPA activity is easily detected colorimetrically in the serum-free supernatant of induced cells (,,). Alternatively, it can be visualized in situ by overlaying colonies of stimulated cells with agar containing both plasminogen and casein (Fig. 1). The degradation of the milk-protein casein by plasmin leads to the formation of lytic zones that allow the identification of uPA secreting colonies. Open image in new window Fig. 1. uPA activity in the casein overlay: Cell lines were induced for 18 h with isoproterenol or calcitonin and subsequently overlaid with a plasminogen and casein containing agar as described in Sections 2. and 3. a,b,c: LLC-PK1 cells; d,e,f: M18 cells; g,h,i: LLC-PK1β3 cells; k,l,m: M18P2.1 cells. a,d,g,k: no induction; b,e,h,l: calcitonin (30 nM); c,f,i,m: isoproterenol (10−5 M). LLC-PK1 β3 was screened and isolated using the calorimetric assay. M18β2.1 was cloned using the casein overlay assay.