Effect of phosphorylation on receptor conformation: the metarhodopsin I in equilibrium with metarhodopsin II equilibrium in multiply phosphorylated rhodopsin.
The superfamily of membrane-bound receptors, which function in signal transduction by activating a guanine nucleotide binding protein or G-protein in response to agonist binding, shares a number of structural and mechanistic properties. Among these similarities is downregulation of functional activity via receptor phosphorylation. In this study, the effects of intermediate levels of phosphorylation (greater than or equal to 4 added phosphates per receptor molecule) on receptor conformational equilibria are examined by comparing the photochemical properties of phosphorylated and unphosphorylated rhodopsins which were incorporated separately into 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine vesicles. Postflash spectra reflecting the contributions of metarhodopsins I, II, and III (meta I, meta II, and meta III) were obtained from these samples. Deconvolution of appropriate difference spectra allowed a determination of the concentration of the photointermediates of interest. Meta II is the form of photolyzed rhodopsin which binds and activates the visual G-protein (Gt); thus, its relative abundance at equilibrium and temporal stability are important parameters in determining the efficiency of visual signal transduction. The effects of pH and temperature on the meta I in equilibrium with meta II equilibrium constant (Keq) and the rate of decay of meta II to meta III were examined for the reconstituted phosphorylated and unphosphorylated rhodopsin samples. Keq was essentially unaffected by phosphorylation when measured at pH 7.0 and 8.0 and 20 and 37 degrees C. The decay time (lifetime) of meta II----meta III had a value of approximately 4.7 min in both phosphorylated and unphosphorylated samples.(ABSTRACT TRUNCATED AT 250 WORDS)
Mitchell
D C
DC
Department of Biochemistry, University of Virginia Health Sciences Center, Charlottesville 22908.
Kibelbek
J
J
Litman
B J
BJ
D000818:Animals; D002417:Cattle; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008081:Liposomes; D010713:Phosphatidylcholines; D010766:Phosphorylation; D011487:Protein Conformation; D012243:Rhodopsin / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism*; D012374:Rod Cell Outer Segment / Q000378:metabolism; D015398:Signal Transduction; D013053:Spectrophotometry; D013816:Thermodynamics
D008081:Liposomes; D010713:Phosphatidylcholines; C013110:metarhodopsins; D012243:Rhodopsin; C028694:1-palmitoyl-2-oleoylphosphatidylcholine