Engineering Visual Arrestin-1 with Special Functional Characteristics

Vishnivetskiy, Sergey A.; Chen, Qiuyan; Palazzo, Marco; Brooks, Evan K.; Altenbach, Christian; Iverson, T.M.; Hubbell, Wayne L.; Gurevich, Vsevolod V.

doi:10.1074/jbc.m112.445437

Cited by 31 publications

(56 citation statements)

References 71 publications

(53 reference statements)

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“…Sequence entropies were determined using protein interface evaluation with evolutionary analysis (51). facilitate engineering of arrestin-1 with specific functional characteristics (45,46). Transfer of mutations to arrestin-2 and arrestin-3 sets the stage to develop diagnostic and therapeutic tools to study diseases caused by hyperactivity of other GPCRs.…”

Section: Resultsmentioning

confidence: 99%

Functional map of arrestin-1 at single amino acid resolution

Ostermaier

Peterhans

Jaussi

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Arrestins function as adapter proteins that mediate G proteincoupled receptor (GPCR) desensitization, internalization, and additional rounds of signaling. Here we have compared binding of the GPCR rhodopsin to 403 mutants of arrestin-1 covering its complete sequence. This comprehensive and unbiased mutagenesis approach provides a functional dimension to the crystal structures of inactive, preactivated p44 and phosphopeptide-bound arrestins and will guide our understanding of arrestin-GPCR complexes. The presented functional map quantitatively connects critical interactions in the polar core and along the C tail of arrestin. A series of amino acids (Phe375, Phe377, Phe380, and Arg382) anchor the C tail in a position that blocks binding of the receptor. Interaction of phosphates in the rhodopsin C terminus with Arg29 controls a C-tail exchange mechanism in which the C tail of arrestin is released and exposes several charged amino acids (Lys14, Lys15, Arg18, Lys20, Lys110, and Lys300) for binding of the phosphorylated receptor C terminus. In addition to this arrestin phosphosensor, our data reveal several patches of amino acids in the finger (Gln69 and Asp73-Met75) and the lariat loops (L249-S252 and Y254) that can act as direct binding interfaces. A stretch of amino acids at the edge of the C domain (Trp194-Ser199, Gly337-Gly340, Thr343, and Thr345) could act as membrane anchor, binding interface for a second rhodopsin, or rearrange closer to the central loops upon complex formation. We discuss these interfaces in the context of experimentally guided docking between the crystal structures of arrestin and light-activated rhodopsin.visual system | scanning mutagenesis | membrane receptor | cell signaling | protein engineering T he human genome encodes more than 800 G protein-coupled receptors (GPCRs), which mediate signaling between cells and provide an important link to our environment as the principal receptors for taste, smell, and vision. The visual system with its photoreceptor rhodopsin is an excellent system to understand GPCR signaling, as detailed information exists on the structures and dynamic interactions of the protein constituents (1). G protein-mediated signaling by light-activated rhodopsin is terminated by a process that begins with the phosphorylation of rhodopsin's C terminus by the rhodopsin kinase GRK1. The phosphorylated, light-activated rhodopsin binds then to arrestin-1, which stops signaling by occluding the G protein-binding site. Further cloning efforts yielded two ubiquitously expressed nonvisual arrestins (arrestin-2 and arrestin-3 or β-arrestin-1 and β-arrestin-2) and the cone-specific arrestin-4. It seems clear today that most GPCRs share a common mechanism of signal termination involving receptor phosphorylation and the binding of arrestins. Arrestin-bound receptors may be internalized and degraded, internalized and recycled, and/or initiate G proteinindependent signaling (2).In recent years there has been tremendous progress in the structure determination of active GPCR states includ...

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Section: Resultsmentioning

confidence: 99%

Functional map of arrestin-1 at single amino acid resolution

Ostermaier

Peterhans

Jaussi

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…Rhodopsin Purification and in Vitro Phosphorylation-Rhodopsin (Rho) in native disk membranes was purified from bovine retina as described previously (26). Opsin was generated from rhodopsin, as described previously (27).…”

Section: Methodsmentioning

confidence: 99%

G Protein-coupled Receptor Kinases of the GRK4 Protein Subfamily Phosphorylate Inactive G Protein-coupled Receptors (GPCRs)

Homan

Vishnivetskiy

et al. 2015

Journal of Biological Chemistry

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“…Mutations were introduced by PCR using the strategy previously described (31,32). All constructs were confirmed by dideoxy sequencing.…”

Section: Materials-[␥-mentioning

confidence: 99%

“…Direct binding assay was performed as described (32,33). Briefly, 1 nM arrestin-1 (50 fmol) was incubated with 0.3 g of various functional forms of rhodopsin in 50 l of 50 mM Tris-HCL, pH 7.4, 100 mM potassium acetate, 1 mM EDTA, 1 mM DTT for 5 min at 37°C under room light (P-Rh*, Rh*, or phospho-opsin) or in the dark (P-Rh or Rh).…”

Section: Materials-[␥-mentioning

confidence: 99%

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Critical Role of the Central 139-Loop in Stability and Binding Selectivity of Arrestin-1

Vishnivetskiy

Baameur

Findley

et al. 2013

Journal of Biological Chemistry

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Background: Arrestin-1 selectively binds light-activated phosphorhodopsin. Results: Deletions in the 139-loop or disruptions of its interactions with the body of arrestin-1 greatly reduce arrestin-1 stability and selectivity. Conclusion: The central 139-loop supports basal arrestin-1 conformation and reduces its binding to non-preferred forms of rhodopsin. Significance: The central 139-loop is an earlier unappreciated element contributing to the thermal stability and binding selectivity of arrestins.

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Engineering Visual Arrestin-1 with Special Functional Characteristics

Cited by 31 publications

References 71 publications

Functional map of arrestin-1 at single amino acid resolution

Functional map of arrestin-1 at single amino acid resolution

G Protein-coupled Receptor Kinases of the GRK4 Protein Subfamily Phosphorylate Inactive G Protein-coupled Receptors (GPCRs)

Critical Role of the Central 139-Loop in Stability and Binding Selectivity of Arrestin-1

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