Biochemistry/Molecular Biology - ARVO

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular BiologyCritical role of central 139 loop in stability and binding selectivityof arrestin-1Sergey A. Vishnivetskiy, Faiza Baameur, Kristen R. Findley,Vsevolod V. Gurevich. Pharmacology, Vanderbilt University,Nashville, TN.Purpose: To elucidate the functional role of central 139-loop on thereceptor-binding surface of arrestin-1Methods: Deletions of 139-loop and mutations that disrupt itsinteractions with other elements of the molecule were introduced inwild type arrestin-1 and its enhanced 3A mutant with high binding tounphosphorylated light-activated rhodopsin (Rh*). We tested bindingselectivity and thermal stability of these mutantsResults: Using intra-molecular distance measurements by DEER wedetected large movement of arrestin-1 139-loop to the side, awayfrom the incoming receptor, upon its binding to activephosphorhodopsin (P-Rh*). Therefore, we hypothesized thatelimination of this loop would promote rhodopsin binding. Deletionsof increasing length in 139-loop progressively enhanced arrestin-1binding to non-preferred forms of rhodopsin: Rh*, dark P-Rh, andphospho-opsin. The interactions of 139-loop with the neighboringfinger loop involved in receptor binding were disrupted by chargeeliminations and reversals on both sides. These milder perturbationsincreased arrestin-1 binding to non-preferred forms of rhodopsin, butto a lesser extent. In all cases an increase in binding to Rh*, dark P-Rh, and phospho-opsin correlated with a decrease in thermal stabilityof the mutants. Similar effects of deletions and mutations in 139-loopon both binding selectivity and thermal stability were observed in thecontext of WT arrestin-1 and an enhanced 3A mutant. whichdemonstrates higher binding to non-preferred forms of rhodopsinConclusions: Central 139 loop is an earlier unappreciated elementstabilizing basal arrestin-1 conformation and precluding its binding tonon-preferred forms of rhodopsin. Thermal stability of the basal stateof arrestin-1 directly correlates with its selectivity for P-Rh*. NIHgrants EY011500, GM077561, and GM081756 (VVG).Commercial Relationships: Sergey A. Vishnivetskiy, None; FaizaBaameur, None; Kristen R. Findley, None; Vsevolod V. Gurevich,NoneSupport: NIH grants EY011500, GM077561, and GM081756(VVG).Program Number: 2459 Poster Board Number: D0064Presentation Time: 2:45 PM - 4:30 PMModulation of Mouse Rod cGMP-Gated Channels by Grb14Raju V. Rajala 1 , Michael L. Woodruff 2 , Gordon L. Fain 2, 3 .1 Ophthal/Dean McGee Eye Inst, Univ of Oklahoma Hlth Sci Ctr,Oklahoma City, OK; 2 Integrative Biology and Physiology, UCLA,Los Angeles, CA; 3 Jules Stein Eye Inst, UCLA, Los Angeles, CA.Purpose: Previous experiments indicated that growth factor receptorboundprotein 14 (Grb14) may modulate rod cyclic guanosinenucleotide (cGMP) gated channels by decreasing channel affinity forcGMP. We tested this hypothesis by recording electrical responsesfrom rods in which the gene for the Grb14 protein had been deleted.Methods: Grb14 -/- mice were obtained from Dr. Roger Daly, GarvanInstitute of Medical Research, Australia. Suction-electrode recordingswere made from single mouse rods by methods previously described(e.g. Chen et al., J. Neurosci. 30:16232-40, 2010).Results: Rod responses of dark-adapted Grb14 -/- mice decayed morerapidly than strain-controlled wild-type (WT) rods (Figure 1) withdecreased values of τ REC at all light intensities; integration timedecreased from 292 ± 20 ms (SE, n=15) in WT to 215 ± 12 ms (17)in Grb14 -/- mice. This result is consistent with an increase in channelaffinity for cGMP produced by deletion of Grb14. Grb14 -/- mouserods also showed a large and significant decrease in the limiting timeconstant τ D from 171 ± 15 ms (15) in WT to 118 ± 5 ms (17) inGrb14 -/- rods. Similar though smaller decreases in both τ REC and τ Dwere previously reported from mice whose channels lack thecalmodulin binding site (Chen et al., 2010), which should also resultin increased channel affinity for cGMP. Although Grb14 wasreported to translocate from inner to outer segment in the light(Rajala et al. Biochemistry 48:5563-5572, 2009), we saw no apparentdifference in the effect of Grb14 gene deletion on response decay indark-adapted and bleached or background adapted rods, and lightadaption in Grb14 -/- rods was essentially the same as in WT.Conclusions: Our results confirm a role of Grb14 as a modulator ofmouse rod cGMP-gated channels in both dark-adapted and lightadaptedrods. The physiological role of this modulation remains to beelucidated.Figure 1. Responses of WT and Grb14 -/- rods to 10 ms flashes ofintensities from 2.4 to 2600 photons μm -2 .Commercial Relationships: Raju V. Rajala, None; Michael L.Woodruff, None; Gordon L. Fain, NoneSupport: NIH/NEI grant (EY01844, EY016507, EY00871)Program Number: 2460 Poster Board Number: D0065Presentation Time: 2:45 PM - 4:30 PMIndependent manipulation of binding selectivity and selfassociationof arrestin-1Vsevolod V. Gurevich 1 , Sergey A. Vishnivetskiy 1 , Qiuyan Chen 1 ,Maria C. Palazzo 1 , Evan K. Brooks 2 , Christian Altenbach 2 , Tina M.Iverson 1 , Wayne L. Hubbell 2 . 1 Pharmacology, Vanderbilt University,Nashville, TN; 2 JSEI, UCLA, Los Angeles, TN.Purpose: To test whether binding selectivity and self-association ofarrestin-1 can be independently manipulated for research andtherapeutic useMethods: Versions of arrestin-1 with activating mutations enhancingthe binding to unphosphorylated light-activated rhodopsin (Rh*) andsubstitutions suppressing self-association were constructed,expressed, and their binding selectivity, stability, and oligomerizationwere testedResults: Arrestin-1 preferentially binds active phosphorylatedrhodopsin (P-Rh*). An enhanced mutant with increased binding toRh* partially compensates for the lack of rhodopsin phosphorylationin vivo. We show that reengineering of the receptor-binding surfaceof arrestin-1 further improves the binding to Rh* while preservingprotein stability. Mammalian arrestin-1 readily self-associates atphysiological concentrations. To elucidate the biological role of thisphenomenon, wild type arrestin-1 in living animals must be replacedwith a non-oligomerizing mutant retaining all other functions.©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.