Analysis of dimerization determinants of PDE6 catalytic subunits.
An absolute majority of cyclic nucleotide phosphodiesterases (PDEs) form catalytic dimers. The structural determinants and functional significance of PDE dimerization are poorly understood. Furthermore, all known dimeric PDEs with the exception of retinal rod guanosine 3',5'-cyclic-monophosphate PDE (PDE6) are homodimeric enzymes. Rod PDE6 is a catalytic heterodimer composed of alpha- and beta-subunits. Gel filtration, sucrose gradient centrifugation, and immunoprecipitation are standard techniques used to study dimerization of proteins. We successfully applied these methods to investigate dimerization of chimeric proteins between PDE6alphabeta and PDE5, which allowed us to elucidate the structural basis for heterodimerization of rod PDE6. This chapter outlines approaches to the investigation of PDE6 dimerization that can be utilized in a broader analysis of PDE dimerization.
Muradov
Khakim G
KG
Department of Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, USA.
Boyd
Kimberly K
KK
Artemyev
Nikolai O
NO
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000737:chemistry*; D000818:Animals; D002499:Centrifugation, Density Gradient / Q000379:methods; D002850:Chromatography, Gel / Q000379:methods; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5; D054707:Cyclic Nucleotide Phosphodiesterases, Type 6; D019281:Dimerization; D005136:Eye Proteins; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods
D005136:Eye Proteins; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5; D054707:Cyclic Nucleotide Phosphodiesterases, Type 6; C517381:PDE5A protein, human; C410522:PDE6A protein, human; C517431:PDE6B protein, human