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V V Gurevich

Publications and source records attributed to V V Gurevich.

50 records · Page 3Linked to original sources
PubMed1994

Arrestin-rhodopsin interaction. Multi-site binding delineated by peptide inhibition.

Visual arrestin modulates the intracellular response of retinal rod cells to light by specifically binding to the phosphorylated light-activated form of the photoreceptor rhodopsin (P-Rh*). In order to characterize the molecular interaction between rhodopsin and arrestin, we have studied the ability of synthetic peptides from the proposed cytoplasmic loops of rhodopsin to inhibit arrestin binding. A third cytoplasmic loop peptide competed most effectively for arrestin binding to P-Rh*, exhibiting an IC50 of 34 microM, while a first cytoplasmic loop peptide weakly inhibited binding with an IC50 of approximately 1100 microM. The first and third cytoplasmic loop peptides also inhibited P-Rh* interaction with both ARR[delta (2-16)-404], an arrestin mutant that lacks residues 2-16, and ARR[1-191], a mutant that contains only the amino half of arrestin. However, the third loop peptide had an approximately 5-fold lower affinity at inhibiting the binding of ARR[1-191] to P-Rh*. While the first and third loop peptides also inhibited arrestin binding to light-activated rhodopsin and a truncated rhodopsin lacking its C-terminal sites of phosphorylation, the peptides modestly enhanced arrestin binding to phosphorylated dark rhodopsin. These results suggest that the third and, to a lesser extent, the first cytoplasmic loops of rhodopsin may play an important role in arrestin binding to light-activated forms of rhodopsin.

Amino Acid Sequence↗
PubMed1993

Binding of wild type and chimeric arrestins to the m2 muscarinic cholinergic receptor.

Arrestins play an important role in regulating the activity of the G protein-coupled receptors rhodopsin and the beta 2-adrenergic receptor. Recently, we described the expression and functional characterization of visual arrestin using an in vitro translation system. Here we report the expression of beta-arrestin and development of a direct binding assay to study the interaction of arrestins with a muscarinic cholinergic receptor. In vitro translated beta-arrestin was found to specifically bind to purified reconstituted human m2 muscarinic cholinergic receptor (hm2 mAChR) in an agonist- and phosphorylation-dependent manner. Visual arrestin also bound to the hm2 mAChR, albeit to a lesser extent and with lower affinity. In an attempt to dissect the major domains responsible for determining the receptor binding specificity of arrestin and beta-arrestin, we generated several chimeric arrestins. One contained the first 340 residues of beta-arrestin followed by residues 346-404 of arrestin (BRV4), another consisted of the first 207 residues of beta-arrestin and residues 214-404 of visual arrestin (BV3), and a third had residues 1-43 of beta-arrestin replaced by residues 1-47 of arrestin (VIN1). All of these arrestins were able to specifically bind to the activated and phosphorylated form of both the hm2 mAChR and rhodopsin, with a clear preference for the muscarinic receptor. The Kd values for beta-arrestin, BRV4, BV3, VIN1, and visual arrestin binding to the hm2 mAChR were 0.48 +/- 0.06, 0.51 +/- 0.19, 1.38 +/- 0.26, 1.13 +/- 0.26, and 7.2 +/- 1.2 nM, respectively. These data demonstrate that: 1) beta-arrestin binds to the hm2 mAChR in an activation- and phosphorylation-dependent fashion, 2) visual arrestin has 15-fold lower affinity for the hm2 mAChR as compared to beta-arrestin, and 3) the N-terminal half of beta-arrestin plays a key role in determining receptor binding specificity. The use of in vitro translated arrestins to directly assess receptor binding may serve as a viable approach for elucidating the specificity and molecular mechanisms involved in receptor-arrestin interaction.

Animals↗
PubMed1993

Polypeptide variants of beta-arrestin and arrestin3.

Retinal arrestin (S-antigen) inactivates the phototransduction cascade by binding to light-activated phosphorylated rhodopsin and thereby "arresting" coupling to the G protein transducin. beta-Arrestin (beta arr), a ubiquitous arrestin homolog, acts analogously to desensitize the beta 2-adrenergic receptor by disrupting Gs receptor interaction. In an attempt to identify additional "arrestins" which might regulate the multitude of G protein-coupled receptors, we have isolated two bovine brain cDNAs which encode polypeptide variants of an arrestin homolog which we have designated arrestin3 (arr3). The open reading frames of these two cDNAs are identical except that the long form, arr3L, contains an 11-amino-acid insert between residues 361 and 362. Arr3 is more closely related to bovine beta arr (78% identity) than to bovine visual arrestin (56% identity). Polymerase chain reaction amplification of RNA and immunoblotting of lysates with an arr3-specific antibody suggest that the short form, arr3S, is the major form of arr3 in all bovine tissues and that it is most abundant in the spleen. Furthermore, polymerase chain reaction amplification of beta arr mRNA indicates that in several tissues (lung, liver, spleen, and pituitary), the major form of beta arr lacks 8 amino acids which are present in brain beta arr. Immunoblotting with an antibody which recognizes beta arr and arr3 with equal sensitivity demonstrates that beta arr (either the long or the short polypeptide) is the major arrestin in all (non-photoreceptor bearing) tissues examined. These observations suggest that in some tissues, as many as four arrestin homolog variants may play a role in the regulation of G protein-coupled receptors.

Amino Acid Sequence↗
PubMed1993

Visual arrestin interaction with rhodopsin. Sequential multisite binding ensures strict selectivity toward light-activated phosphorylated rhodopsin.

Visual arrestin plays an important role in regulating light responsiveness via its ability to specifically bind to the phosphorylated and light-activated form of rhodopsin. Previously, we utilized an in vitro translation system to express and characterize the full-length (404 amino acids) and two truncated forms of visual arrestin. Here we have extended these studies to include a total of 33 different truncation and deletion mutants of arrestin, ranging from 69 to 391 amino acids in length. Mutants were produced by cutting within the open reading frame of the bovine arrestin cDNA with selective restriction enzymes followed by in vitro translation of the transcribed truncated mRNAs. Mutant arrestin binding to dark, light-activated, dark phosphorylated, and light-activated phosphorylated rhodopsin as well as to opsin, phosphoopsin, and truncated rhodopsin was then extensively characterized. In addition, the sensitivity of arrestin/rhodopsin interactions to conditions of increasing ionic strength was measured. These studies suggest the localization of multiple functional domains within the arrestin molecule that include: 1) a "phosphorylation recognition" domain, which interacts with the phosphorylated carboxyl terminus of rhodopsin, was localized predominately between residues 158-185; 2) an "activation recognition" domain, which interacts with those portions of the rhodopsin molecule that change conformation upon light activation, was found to consist of at least three regions within the first 191 residues of the arrestin molecule; 3) a hydrophobic interaction domain, localized between residues 191 and 365, appears to be mobilized upon binding of arrestin to activated phosphorylated rhodopsin; 4) a regulatory domain, localized in the COOH-terminal region of arrestin (residues 365-391), was found to play a role in controlling the conformational change in arrestin necessary for mobilization of the hydrophobic interaction domain; and 5) The NH2 terminus of arrestin (residues 2-16) was found to be important for interacting with the regulatory COOH-terminal region as well as maintaining the conformation of the NH2-terminal half of arrestin. A mechanism which ensures strict arrestin binding selectivity toward phosphorylated light-activated rhodopsin is proposed.

Amino Acid Sequence↗
PubMed1992

Cell-free expression of visual arrestin. Truncation mutagenesis identifies multiple domains involved in rhodopsin interaction.

Visual arrestin plays an important role in regulating light responsiveness via its ability to specifically bind to the phosphorylated and light-activated form of rhodopsin. To further characterize rhodopsin/arrestin interactions we have utilized a rabbit reticulocyte lysate translation system to synthesize bovine visual arrestin. The translated arrestin (404 amino acids) was demonstrated to be fully functional in terms of its ability to specifically recognize and bind to phosphorylated light-activated rhodopsin (P-Rh*). Competitive binding studies revealed that the in vitro synthesized arrestin and purified bovine visual arrestin had comparable affinities for P-Rh*. In an effort to assess the functional role of different regions of the arrestin molecule, two truncated arrestin mutants were produced by cutting within the open reading frame of the bovine arrestin cDNA with selective restriction enzymes. In vitro translation of the transcribed truncated mRNAs resulted in the production of arrestins truncated from the carboxyl terminus. The ability of each of the mutant arrestins to bind to dark (Rh), light-activated (Rh*), dark phosphorylated (P-Rh), and light-activated phosphorylated rhodopsin were then compared. Arrestin lacking 39 carboxyl-terminal residues binds specifically not only to P-Rh* but also to Rh* and P-Rh. This suggests that the carboxyl-terminal domain of arrestin plays an important regulatory role in ensuring strict arrestin binding selectivity to P-Rh*. Arrestin that has only the first 191 amino-terminal residues predominately discriminates the phosphorylation state of the rhodopsin; however, it also retains some binding specificity for the activation state. These results suggest that the amino-terminal half of arrestin contains key rhodopsin recognition sites responsible for interaction with both the phosphorylated and light-activated forms of rhodopsin.

Animals↗
PubMed1991

Preparative in vitro mRNA synthesis using SP6 and T7 RNA polymerases.

A method for preparative in vitro mRNA synthesis is presented. The method makes it possible to generate several hundred RNA copies per molecule of linearized DNA template. The protocol is applicable to the synthesis of RNAs of differing lengths (from 200 to 3000 bases), and both SP6 and T7 RNA polymerases can be used. The transcription can be scaled up to at least 6-12 ml, yielding 4.5-18 mg of homogeneous biologically active mRNA.

DNA-Directed RNA Polymerases↗
PubMed1991

Proper cotranslational insertion of visual rhodopsin into the lipid bilayer occurs in the absence of protein translocation machinery.

The insertion of visual opsin into membranes occurred during in vitro translation of opsin mRNA in wheat germ extract in the presence of either microsomes or liposomes. The rhodopsin that integrated into both types of membranes after regeneration with 11-cis-retinal was functionally active (in contrast to the nonincorporated protein). Opsin either cotranslationally translocated into microsomes or inserted into liposomes had equal sensitivity to proteolysis and yielded the same pattern of peptides, which differed substantially from the set of peptides produced during proteolysis of opsin not incorporated into membranes. Thus visual opsin does not require protein translocation machinery for proper insertion into the lipid bilayer.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1990

Functional expression in vitro of bovine visual rhodopsin.

Expression of functionally active bovine visual rhodopsin was achieved by sequential transcription and translation in vitro of rhodopsin gene cDNA with co-translational insertion of the protein into phosphatidylcholine liposomes. The recombinant rhodopsin has functional, spectral and immunochemical properties similar to those of natural rhodopsin from bovine retina. Two mutant rhodopsins, Cys316----Ser and Asp330----Asn, Asp331----Asn, were produced by oligonucleotide-directed mutagenesis. The first mutation does not affect rhodopsin's ability to stimulate transducin GTPase and visual cGMP phosphodiesterase activities, while the second double mutation leads to a sharp decrease in rhodopsin activity.

Animals↗
PubMed1990

Bovine rhodopsin: amino acid substitutions Asp-83----Asn and Glu-134----Gln prevent activation of cyclic GMP phosphodiesterase.

Two bovine rhodopsin mutants with substitutions of negatively charged residues within transmembrane domains II and III by uncharged ones (Asp-83----Asn and Glu-134----Gln) were constructed. Both mutants stimulated transducin GTPase with slightly lowered efficiency, but were completely unable to activate cyclic GMP phosphodiesterase.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1990

[Synthesis of visual rhodopsin in a cell-free translation system. II. Functional properties of recombinant rhodopsin and its mutant forms].

Functional expression of bovine visual rhodopsin in the cell-free translation system with cotranslational insertion of the protein into phosphatidylcholine liposomes is described. The recombinant rhodopsin has spectral and functional properties similar to those of natural rhodopsin from bovine retina. Two mutant rhodopsins with amino acid substitutions in the hydrophilic C-terminal domain were obtained using oligonucleotide-directed mutagenesis. It was found that substitution Cys-316----Ser does not affect rhodopsin's ability to activate the visual amplification cascade, whereas double mutation Asp-330----Asn, Asp-331----Asn dramatically lowers the rhodopsin functional activity.

Amino Acid Sequence↗
PubMed1988

[Synthesis of visual rhodopsin in a cell-free translation system. I. Influence of the structure of the synthetic bovine visual opsin mRNA on its translational efficiency].

Influence of structural changes in nontranslated regions and translation initiation site of the in vitro synthesized bovine opsin mRNA on its translational efficiency in the wheat germ cell-free system has been studied. It is shown that level of the opsin synthesis up to 30 micrograms per 1 ml of translational mixture can be attained by optimizing structure of 5'-nontranslated region.

Animals↗
PubMedDate not supplied

[Effect of verapamil (isoptin) on kidney function].

In experiments on conscious dogs intravenous administration of verapamil (3-4 mg/kg) reduced diuresis, natriuresis and glomerular filtration. After that the filtration returned to normal and diuresis and natriuresis increased. Following infusion into the renal artery verapamil enhanced diuresis and natriuresis because of the inhibition of tubular reabsorption. In rats verapamil (5-10 mg/kg) also induced a rise in diuresis and sodium excretion, but water diuresis is reduced at first.

Animals↗