ROD PHOTORECEPTOR CGMP-PHOSPHODIESTERASE - ANALYSIS OF ALPHA-SUBUNITSAND BETA-SUBUNITS EXPRESSED IN HUMAN KIDNEY-CELLS

The bovine alpha and murine beta subunits of rod-photoreceptor cGMP-ph osphodiesterase (PDE(alpha) and PDE(beta)) were expressed in adenoviru s-transformed 293 human embryonic kidney cells. RNA blots from transfe cted cells showed transcripts of 3.0 and 2.8 kb corresponding to PDE(a lpha) and PDE(beta), respectively. Protein expression was analyzed by using affinity-purified antibodies against cGMP-PDE on immunoblots and by immunoprecipitation. PDE(alpha) and PDE(beta) exhibited the expect ed mobility (and thus apparent molecular size) and had cGMP hydrolytic activity. Reconstitution of the PDE alphabeta heterodimer with the ex pressed proteins increased by almost-equal-to 6-fold the activity of t he individual alpha and beta subunits. Addition of expressed beta subu nit to retinal extracts from 9- to 10-day-old rd/rd mice (which have o nly normal alpha and gamma subunits of rod cGMP-PDE and thus minimal a ctivity) increased enzyme activity by almost-equal-to 3-fold. Our resu lts therefore demonstrate that photoreceptor-specific cGMP-PDE can be synthesized in human kidney cells with consequent expression of enzyma tic activity.