Photoreceptor cGMP phosphodiesterase delta subunit (PDEdelta) functions as a prenyl-binding protein.
Bovine PDEdelta was originally copurified with rod cGMP phosphodiesterase (PDE) and shown to interact with prenylated, carboxymethylated C-terminal Cys residues. Other studies showed that PDEdelta can interact with several small GTPases including Rab13, Ras, Rap, and Rho6, all of which are prenylated, as well as the N-terminal portion of retinitis pigmentosa GTPase regulator and Arl2/Arl3, which are not prenylated. We show by immunocytochemistry with a PDEdelta-specific antibody that PDEdelta is present in rods and cones. We find by yeast two-hybrid screening with a PDEdelta bait that it can interact with farnesylated rhodopsin kinase (GRK1) and that prenylation is essential for this interaction. In vitro binding assays indicate that both recombinant farnesylated GRK1 and geranylgeranylated GRK7 co-precipitate with a glutathione S-transferase-PDEdelta fusion protein. Using fluorescence resonance energy transfer techniques exploiting the intrinsic tryptophan fluorescence of PDEdelta and dansylated prenyl cysteines as fluorescent ligands, we show that PDEdelta specifically binds geranylgeranyl and farnesyl moieties with a Kd of 19.06 and 0.70 microm, respectively. Our experiments establish that PDEdelta functions as a prenyl-binding protein interacting with multiple prenylated proteins.
Zhang
Houbin
H
Department of Neurobiology and Anatomy, University of Utah, Salt Lake City, Utah 84112, USA.
Liu
Xiao-hui
XH
Zhang
Kai
K
Chen
Ching-Kang
CK
Frederick
Jeanne M
JM
Prestwich
Glenn D
GD
Baehr
Wolfgang
W
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D054707:Cyclic Nucleotide Phosphodiesterases, Type 6; D003545:Cysteine; D031541:Fluorescence Resonance Energy Transfer; D020558:GTP Phosphohydrolases / Q000378:metabolism; D007150:Immunohistochemistry; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D020419:Photoreceptor Cells, Vertebrate / Q000201:enzymology*; D017368:Protein Prenylation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017948:Retinal Rod Photoreceptor Cells / Q000166:cytology / Q000201:enzymology*
D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D054707:Cyclic Nucleotide Phosphodiesterases, Type 6; D020558:GTP Phosphohydrolases; D003545:Cysteine