Mechanism of rhodopsin kinase regulation by recoverin.
Recoverin is suggested to inhibit rhodopsin kinase (GRK1) at high [Ca(2+)] in the dark state of the photoreceptor cell. Decreasing [Ca(2+)] terminates inhibition and facilitates phosphorylation of illuminated rhodopsin (Rh*). When recoverin formed a complex with GRK1, it did not interfere with the phosphorylation of a C-terminal peptide of rhodopsin (S338-A348) by GRK1. Furthermore, while GRK1 competed with transducin on interaction with rhodopsin and thereby suppressed GTPase activity of transducin, recoverin in the complex with GRK1 did not influence this competition. Constructs of GRK1 that encompass its N-terminal, catalytic or C-terminal domains were used in pull-down assays and surface plasmon resonance analysis to monitor interaction. Ca(2+)-recoverin bound to the N-terminus of GRK1, but did not bind to the other constructs. GRK1 interacted with rhodopsin also by its N-terminus in a light-dependent manner. No interaction was observed with the C-terminus. We conclude that inhibition of GRK1 by recoverin is not the result of their direct competition for the same docking site on Rh*, although the interaction sites of GRK1/Rh* and GRK1/recoverin partially overlap. The N-terminus of GRK1 is recognized by Rh* leading to a conformational change which moves the C-terminus of Rh* into the catalytic kinase groove. Ca(2+)-recoverin interacting with the N-terminus of GRK1 prevents this conformational change and thus blocks Rh* phosphorylation by GRK1.
Komolov
Konstantin E
KE
Biochemistry group, Faculty V, IBU, University of Oldenburg, Oldenburg, Germany.
Senin
Ivan I
II
Kovaleva
Nadezda A
NA
Christoph
Mathias P
MP
Churumova
Valeriya A
VA
Grigoriev
Ilya I
II
Akhtar
Muhammad
M
Philippov
Pavel P
PP
Koch
Karl-Wilhelm
KW
D000494:Allosteric Regulation / Q000502:physiology; D000818:Animals; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000502:physiology; D020134:Catalytic Domain / Q000502:physiology; D002417:Cattle; D051606:G-Protein-Coupled Receptor Kinase 1 / Q000737:chemistry / Q000378:metabolism*; D020419:Photoreceptor Cells, Vertebrate / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D011487:Protein Conformation; D017434:Protein Structure, Tertiary / Q000502:physiology; D051603:Recoverin / Q000737:chemistry / Q000378:metabolism*; D012243:Rhodopsin / Q000737:chemistry / Q000378:metabolism*; D014785:Vision, Ocular / Q000502:physiology*
D051603:Recoverin; D012243:Rhodopsin; D051606:G-Protein-Coupled Receptor Kinase 1; D002118:Calcium