Complementary interactions of the rod PDE6 inhibitory subunit with the catalytic subunits and transducin.
Activation of the cyclic GMP phosphodiesterase (PDE6) by transducin is the central event of visual signal transduction. How the PDE6 inhibitory gamma-subunit (Pgamma) interacts with the catalytic subunits (Palphabeta) and the transducin alpha-subunit (alpha(t)) in this process is not entirely clear. Here we have investigated this issue, taking advantage of site-specific label transfer from throughout the full-length Pgamma molecule to both alpha(t) and Palphabeta. The interaction profiling and pull-down experiments revealed that the Pgamma C- terminal domain accounted for the major interaction with alpha(t) bound with guanosine 5'-3-O-(thio)triphosphate (alpha(t)GTPgammaS) in comparison with the central region, whereas an opposite pattern was observed for the Pgamma-Palphabeta interaction. This complementary feature was further exhibited when both alpha(t)GTPgammaS and Palphabeta were present and competing for Pgamma interaction, with the Pgamma C-terminal domain favoring alpha(t), whereas the central region demonstrated a preference for Palphabeta. Furthermore, alpha(t)GTPgammaS co-immunoprecipitated with PDE6 and vice versa in a Pgamma-dependent manner. Either Palphabeta or alpha(t)GTPgammaS could be pulled down by the Btn-Pgamma molecules on streptavidin beads that were saturated by the other partner, indicating simultaneous binding of these two partners to Pgamma. These data together indicate that complementary Pgamma interactions with its two targets facilitate the alpha(t).PDE6 "transducisome" formation. Thus, our study provides new insights into the molecular mechanisms of PDE6 activation.
Department of Pharmacology, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin 53706. Electronic address: lianwangguo@wisc.edu.
Lian-Wang
LW
Guo
Department of Pharmacology, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin 53706; Pharmaceutical Laboratory, College of Chemistry, Isfahan University of Technology, Isfahan 84156, Iran.
Abdol R
AR
Hajipour
Department of Pharmacology, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin 53706.
Arnold E
AE
Ruoho
D000818:Animals; D020134:Catalytic Domain; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D054707:Cyclic Nucleotide Phosphodiesterases, Type 6 / Q000037:antagonists & inhibitors* / Q000737:chemistry; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D047468:Immunoprecipitation; D017948:Retinal Rod Photoreceptor Cells / Q000201:enzymology*; D015293:Transducin / Q000737:chemistry / Q000378:metabolism*
D016244:Guanosine 5'-O-(3-Thiotriphosphate); D054707:Cyclic Nucleotide Phosphodiesterases, Type 6; D015293:Transducin