COMMUNICATION
Targeted Construction of Phosphorylation-independent beta -Arrestin Mutants with Constitutive Activity in Cells*

Abraham KovoorDagger §, Jeremy CelverDagger §, Ravil I. Abdryashitov, Charles ChavkinDagger , and Vsevolod V. Gurevichparallel

From the  Ralph & Muriel Roberts Laboratory for Vision Science, Sun Health Research Institute, Sun City, Arizona 85372 and the Dagger  Department of Pharmacology, University of Washington, Seattle, Washington 98195-7280

    ABSTRACT
Top
Abstract
Introduction
References

Arrestin proteins play a key role in the desensitization of G protein-coupled receptors (GPCRs). Recently we proposed a molecular mechanism whereby arrestin preferentially binds to the activated and phosphorylated form of its cognate GPCR. To test the model, we introduced two different types of mutations into beta -arrestin that were expected to disrupt two crucial elements that make beta -arrestin binding to receptors phosphorylation-dependent. We found that two beta -arrestin mutants (Arg169 right-arrow Glu and Asp383 right-arrow Ter) (Ter, stop codon) are indeed "constitutively active." In vitro these mutants bind to the agonist-activated beta 2-adrenergic receptor (beta 2AR) regardless of its phosphorylation status. When expressed in Xenopus oocytes these beta -arrestin mutants effectively desensitize beta 2AR in a phosphorylation-independent manner. Constitutively active beta -arrestin mutants also effectively desensitize delta  opioid receptor (DOR) and restore the agonist-induced desensitization of a truncated DOR lacking the critical G protein-coupled receptor kinase (GRK) phosphorylation sites. The kinetics of the desensitization induced by phosphorylation-independent mutants in the absence of receptor phosphorylation appears identical to that induced by wild type beta -arrestin + GRK3. Either of the mutations could have occurred naturally and made receptor kinases redundant, raising the question of why a more complex two-step mechanism (receptor phosphorylation followed by arrestin binding) is universally used.

    INTRODUCTION
Top
Abstract
Introduction
References

The decrease of a response to a persistent stimulus (desensitization) is a widespread biological phenomenon. Signaling by diverse G protein-coupled receptors (GPCRs)1 is believed to be terminated by a uniform two-step mechanism (1). According to the model, activated receptor is first phosphorylated by a G protein-coupled receptor kinase (GRK). An arrestin protein binds to the activated phosphoreceptor, thereby blocking G protein interaction. Arrestin-receptor complex is then internalized, whereupon receptor is either dephosphorylated and recycled back to the plasma membrane (resensitization) or sorted to lysosomes and destroyed (down-regulation). Thus, the formation of the arrestin-receptor complex appears to be the final step of desensitization and the first step of resensitization and/or receptor down-regulation, which puts it at the crucial cross-roads of the processes regulating cellular responsiveness. The tremendously diverse superfamily of G protein-coupled receptors with more than 1000 members is the largest known group of proteins that translate a wide variety of external stimuli into intracellular "language." In contrast, the repertoire of receptor kinases and arrestins involved in the desensitization of these receptors is rather limited: only six GRKs and four arrestins have thus far been found in mammals (reviewed in Ref. 1). This suggests that at least some of the kinases and arrestins regulate numerous receptors. Thus, these proteins are attractive targets for research designed to delineate common molecular mechanisms underlying the regulation of GPCR signaling in cells (and to create fairly universal tools for the experimental and/or therapeutic intervention in the process).

    EXPERIMENTAL PROCEDURES

Mutagenesis and Biochemical Characterization of beta -Arrestins-- Mutations Arg169 right-arrow Glu (CGG right-arrow GAG), Gln394 right-arrow Ter (CAA right-arrow TAA), and Asp383->Ter (GAT right-arrow TAG) were introduced by polymerase chain reaction in beta -arrestin construct pBARR (3), that was used for in vitro transcription and translation, as described (3). NcoI/HindIII 1404-base pair open reading frame was then subcloned into appropriately digested Escherichia coli expression vector pTrcHisB (Invitrogen). All beta -arrestin species were expressed in the in vitro translation system and tested in the direct binding assay (3), overexpressed in E. coli, purified to apparent homogeneity (16), and characterized in the agonist affinity shift assay (7), essentially as described.

Direct Binding Assay-- In vitro translated tritiated arrestins (50 fmol) were incubated in 50 mM Tris-HCl, pH 7.5, 0.5 mM MgCl2, 1.5 mM dithiothreitol, 50 mM potassium acetate with 7.5 pmol of the various functional forms of rhodopsin or with P-beta 2AR or beta 2AR (100 fmol/assay) in a final volume of 50 µl for 5 min at 37 °C in room light (rhodopsin) or for 60 min at 30 °C in the presence of 0.1 mM beta -agonist isoproterenol. The samples were immediately cooled on ice and loaded onto 2 ml Sepharose 2B columns equilibrated with 20 mM Tris-HCl, pH 7.5, 2 mM EDTA. Bound arrestin eluted with receptor-containing membranes in the void volume (between 0.5 and 1.1 ml). Nonspecific binding determined in the presence of 0.3 µg of liposomes was subtracted.

Agonist Affinity Shift Assay-- P-beta 2AR or beta 2AR (10-15 fmol/assay) was incubated in 0.25 ml of 10 mM Tris-HCl, pH 7.4, 100 mM NaCl (buffer A) containing 0.1 mg/ml bovine serum albumin in the presence of 65-75 fmol of [125I]iodopindolol (NEN Life Science Products) and the indicated concentrations of arrestins and agonists for 60 min at 22 °C. Samples were then cooled on ice and loaded at 4 °C onto 2 ml of Sephadex G-50 columns. Receptor-containing liposomes with bound radioligand were eluted with buffer A (between 0.6 and 1.5 ml), and radioactivity was quantitated in a liquid scintillation counter. Nonspecific binding was determined in the presence of 10 µM alprenolol. All experiments were repeated two to three times, and data are presented as means ± S.D.

Desensitization Studies in Xenopus Oocytes-- Stage IV oocytes from mature female Xenopus laevis frogs were harvested, defolliculated, and cultured as described previously (8). cRNA was prepared for oocyte injection from cDNA template using Ambion message machine kit (Ambion, TX) according to manufacturer's protocol. cDNAs (GenBankTM accession numbers in parentheses) for rat GRK3 (AA144588), human beta 2AR (AI052644), mouse delta  opioid receptor (L06322), and rat G protein-gated inwardly rectifying potassium channel subunits Kir3.1 (U01071) and Kir3.4 (X83584) were amplified and linearized prior to cRNA synthesis. cDNAs for all forms of beta -arrestin were first amplified by polymerase chain reaction using oligonucleotides designed to add a T7 promoter upstream and a 45-base poly(A) tail downstream of beta -arrestin open reading frame. Standard two-electrode voltage clamp recordings were performed to register Kir3 currents activated by agonist perfusion as described (8). The expression levels in oocytes of all forms of beta -arrestin were determined by quantitative Western blot with F4C1 anti-arrestin antibody (22), as described (16). Means ± S.D. from four to six measurements are presented.

    RESULTS AND DISCUSSION

Recently we have proposed a molecular mechanism that explains an amazing selectivity of arrestins for the activated phosphorylated forms of GPCRs (2, 3). According to previous in vitro studies (2, 3) arrestins have two primary binding sites: an activation-recognition site that recognizes the agonist-activated state of the receptor and a phosphorylation-recognition site that interacts with GRK-phosphorylated elements of the receptor. A potent secondary receptor-binding site is mobilized for the interaction only when both primary sites are simultaneously engaged, i.e. when an arrestin encounters activated and phosphorylated receptor (2, 3). In this model, arrestin is kept in its basal conformation by several intramolecular interactions in which certain residues in the primary binding sites ("trigger" residues) are involved. One of these triggers is pulled by binding to an activated form of the receptor, the other, by the interaction of arrestin with phosphate(s) introduced by GRK. Thus, arrestin works like a coincidence detector, assuming its high-affinity receptor-binding conformation when both triggers are simultaneously pulled. The model of sequential multisite binding (2, 3), and the recent crystal structure of visual arrestin (4) set the stage for the targeted construction of arrestin mutants in which one of the triggers is constitutively pulled by an appropriate mutation.

In order to test the validity of the model we constructed three beta -arrestin mutants: 1) Arg169 right-arrow Glu, that reverses the charge of the putative phosphorylation-sensitive trigger (Arg169 right-arrow Glu is homologous to the Arg175 right-arrow Glu mutation in visual arrestin, that makes its binding to rhodopsin phosphorylation-independent (5)); 2) Gln394 right-arrow Ter; and 3) Asp383 right-arrow Ter, that delete a part or all of the regulatory arrestin COOH terminus, which keeps arrestin in a basal conformation and suppresses an untimely mobilization of the secondary binding site (6).

First, we tested the ability of these mutants to interact with purified beta 2AR reconstituted into phospholipid vesicles by performing direct binding studies (3) and agonist affinity shift assays (7) in vitro. Wild type beta -arrestin and beta -arrestin-(1-393) bind poorly to activated unphosphorylated receptor (Fig. 1A). In contrast, beta -arrestin-(Arg169 right-arrow Glu) and beta -arrestin-(1-382) demonstrate significantly higher binding to activated unphosphorylated receptor (Fig. 1A). Wild type beta -arrestin and all three mutants readily bind to activated and phosphorylated beta 2AR (Fig. 1A). Thus, beta -arrestin-(Arg169 right-arrow Glu) and beta -arrestin-(1-382) bind to activated beta 2AR in a phosphorylation-independent fashion.


View larger version (28K):
[in this window]
[in a new window]
 
Fig. 1.   Direct binding assay. A, 100 fmol of beta ARK-phosphorylated (P-beta 2AR, 2.7 ± 0.2 mol phosphate/mol receptor) or unphosphorylated purified beta 2AR reconstituted into liposomes was incubated in a 50 µl reaction with 50 fmol of the indicated form of tritiated arrestin (specific activities: 140-160 dpm/fmol) in the presence of 100 µM agonist isoproterenol in 50 mM Tris-HCl, pH 7.5, 50 mM potassium acetate, 0.5 mM MgCl2 for 45 min at 30 °C. B, 0.3 µg of rhodopsin kinase-phosphorylated (P-Rh*, 1.6 ± 0.1 mol of phosphate/mol) or unphosphorylated (Rh*) rhodopsin was incubated with the same set of arrestins under room light for 5 min at 37 °C. The samples were then cooled on ice and loaded at 4 °C onto 2-ml Sepharose 2B columns, equilibrated with 20 mM Tris-HCl, pH 7.5, 2 mM EDTA. Bound tritiated arrestins were eluted with receptor-containing membranes in the void volume (between 0.5 and 1.1 ml), and the radioactivity was quantitated in a liquid scintillation counter. *, p < 0.01, Student's t test, compared with the binding of corresponding wild type arrestin.

Recently (7) we found that arrestin-receptor complex is similar to G protein-receptor complex in two respects: agonists have higher affinity for arrestin-receptor complex than for receptor alone, and only a fraction of the receptors forms such a high agonist affinity complex (HAC) even at saturating concentrations of arrestin. The maximum percentage of the receptor in HAC gives a good estimate of the propensity of a given arrestin protein to bind tightly to the receptor (arrestin competency) (7). Consistent with the direct binding data (Fig. 1A), beta -arrestin-(Arg169 right-arrow Glu) and beta -arrestin-(1-382) induced the formation of HAC by unphosphorylated beta 2AR (22 ± 4% in both cases) (Fig. 2A). In contrast, all forms of beta -arrestin induced the formation of HAC by phosphorylated beta 2AR (P-beta 2AR) (Fig. 2B). The percentage of HAC formed by P-beta 2AR in the presence of saturating (1 µM) concentration of beta -arrestin, beta -arrestin-(Arg169 right-arrow Glu), beta -arrestin-(1-382), and beta -arrestin-(1-393) was 31 ± 6, 52 ± 3, 41 ± 3, and 20 ± 4%, respectively (Fig. 2B). In summary, in both in vitro assays beta -arrestin-(Arg169 right-arrow Glu) and beta -arrestin-(1-382) demonstrate constitutive activity (phosphorylation-independent receptor binding).


View larger version (24K):
[in this window]
[in a new window]
 
Fig. 2.   Agonist affinity shift assay. Competition curves were generated, as follows. Unphosphorylated (A) or phosphorylated (B) beta 2AR (10-15 fmol/assay) was incubated in 0.25 ml of 10 mM Tris-HCl, pH 7.4, 100 mM NaCl, containing 0.1 mg/ml bovine serum albumin in the presence of 65-75 fmol of beta 2AR-antagonist [125I]iodopindolol (IPIN) (NEN Life Science Products) and the indicated concentrations of beta 2AR-agonist isoproterenol for 60 min at 22 °C in the absence (open circle ) or presence of 1 µM beta -arrestin (), beta -arrestin-(Arg169 right-arrow Glu) (black-triangle), beta -arrestin-(1-382) (black-square), or beta -arrestin-(1-393) (black-diamond ). Samples were then cooled on ice and loaded onto 2-ml Sephadex G-50 columns at 4 °C. Receptor-containing liposomes with bound radioligand were eluted with the same buffer (between 0.6 and 1.5 ml), and the radioactivity was quantitated. For the unphosphorylated beta 2AR competition curves in the absence of arrestins and in the presence of wild type and beta -arrestin-(1-393) are monophasic (analysis using Prism 2.0 for Power Macintosh). In the presence of beta -arrestin-(Arg169 right-arrow Glu) and beta -arrestin-(1-382) the curves are biphasic, suggesting the presence of high- and low-affinity sites. With P-beta 2AR competition curves generated in the presence of all forms of beta -arrestin are biphasic, while the curve in the absence of arrestins is monophasic. The analysis shows that in all cases isoproterenol competes for the binding at high-affinity sites with IC50 of 26 ± 6 nM and the low-affinity sites with IC50 of 730 ± 131 nM. The latter is very close to the affinity of beta 2AR alone (656 ± 27 nM). The fraction of the high-affinity binding sites in each experiment is presented in the text. Experiments were performed two to three times in duplicates. Means ± S.D. are presented.

Next we tested whether these beta -arrestin species can functionally desensitize unphosphorylated beta 2AR in living cells. To this end GPCRs were expressed in Xenopus oocytes and the activation of coexpressed G protein-gated inwardly rectifying K+ channel Kir3 was used as a measure of receptor function. Under these conditions, the application of receptor agonists produced a large increase in inwardly rectifying potassium conductance (8). Undetectable levels of endogenous arrestins and GRKs are expressed in these cells (data not shown). As a result, only a very slow response desensitization was evident during prolonged agonist treatment when beta 2AR (or another GPCR) is expressed alone. The rate of desensitization was not significantly increased when the receptor is coexpressed with either GRK alone or arrestin alone. However, a dramatic increase in desensitization rate was observed when both GRK and arrestin were coexpressed with a receptor (8, 9). To compare the relative activity of different forms of beta -arrestin, we expressed beta 2AR with or without GRK3 (also called beta -adrenergic receptor kinase 2 or beta ARK2) in the presence or absence of different forms of beta -arrestin. As shown on Fig. 3 (A and C), both wild type and beta -arrestin-(1-393) facilitated beta 2AR desensitization only when beta ARK2 was present. In contrast, beta -arrestin-(Arg169 right-arrow Glu) and beta -arrestin-(1-382) in the absence of beta ARK2 produced high rates of desensitization similar to that produced by wild type beta -arrestin in the presence of beta ARK2, suggesting that these mutants did induce phosphorylation-independent desensitization of beta 2AR in the cell.


View larger version (29K):
[in this window]
[in a new window]
 
Fig. 3.   GRK-independent functional desensitization of beta 2AR and DOR by beta -arrestin-(Arg169 right-arrow Glu) and beta -arrestin-(1-382). A and C, beta 2AR function was studied in oocytes injected with a mixture of cRNAs for the beta 2AR, the G protein-gated inwardly rectifying K+ channel subunits Kir3.1 and Kir3.4, and Gsalpha , that allows the Gs-coupled beta 2AR to activate the coexpressed channels, as described (8). B and D, DOR was studied in oocytes injected with cRNAs for DOR, Kir3.1, and Kir3.4 (8). As indicated, some oocytes were also coinjected with 8 ng of cRNA for the different forms of beta -arrestin either alone or together with 0.5 ng of beta ARK2 cRNA. All recordings were performed 3-4 days post-injection. Receptor-activated currents were measured in 16 mM K+ buffer (8) at -80 mV holding potential. The agonist-elicited responses were adjusted by base-line subtraction as described (8) and normalized to the peak response. The short vertical lines through the traces indicate when agonist treatment was discontinued and the corresponding antagonist perfusion started. Antagonist perfusion was used to determine the amount of residual receptor response. Calibration scales are the same for each trace (2 min). Representative traces depicting beta 2AR-activated current responses elicited by 1 µM beta -agonist isoproterenol and reversed by 1 µM of beta -antagonist propranolol (A), DOR-activated current responses elicited by 1 µM DOR agonist DPDPE and reversed by 1 µM DOR antagonist naloxone (B), the beta 2AR desensitization rate in each group of oocytes expressed as a multiple of the desensitization rate in oocyte group injected with cRNA for wild type beta -arrestin only (C), and DOR desensitization rates calculated as for beta 2AR (D) are shown. Hatched bars in C and D represent the corresponding control receptor desensitization rates measured in oocytes expressing neither beta ARK2 nor beta -arrestin. *, p < 0.05, Student's t test, compared with desensitization rate of the group coexpressing wild type beta -arrestin only. Each bar represents the mean ± S.E. from 4-16 separate oocytes. beta -arr, beta -arrestin.

In order to test whether the constitutively active forms of beta -arrestin retain the characteristic broad receptor specificity of wild type nonvisual arrestins (9, 10), we performed similar series of experiments with delta  opioid receptor (DOR) (Fig. 3, B and D), which was previously shown to be desensitized following agonist activation in oocytes coexpressing wild type beta -arrestin and beta ARK2 (8). Again, the constitutively active mutants induced DOR desensitization, even in the absence of beta ARK2, suggesting that these mutations do not appreciably change receptor specificity of beta -arrestin (or, rather, lack thereof). It should be noted that wild type visual and beta -arrestin, visual arrestin mutant (Arg175 right-arrow Glu), and beta -arrestin mutants (Arg169 right-arrow Glu), (1-382), and (1-393), readily bind to activated phosphorylated forms of both rhodopsin and beta 2AR (Fig. 1). Visual arrestin mutant (Arg175 right-arrow Glu) also binds to unphosphorylated activated rhodopsin, while beta -arrestin mutants (Arg169 right-arrow Glu) and (1-382) bind to unphosphorylated activated beta 2AR. However, phosphorylation-independent visual arrestin mutant does not bind to unphosphorylated activated beta 2AR and phosphorylation-independent beta -arrestin mutants do not bind to unphosphorylated activated rhodopsin (Fig. 1). Thus, the preference of beta -arrestin for beta 2AR over rhodopsin and that of visual arrestin for rhodopsin over beta 2AR (3) appears, if anything, enhanced by these mutations.

Interestingly, in the presence of beta ARK2 both of the phosphorylation-independent beta -arrestin mutants induced a more rapid receptor desensitization than wild type beta -arrestin (Fig. 3), although the expression levels of all forms of beta -arrestin in oocytes were virtually the same (0.72 ± 0.34, 0.90 ± 0.27, 0.85 ± 0.32, and 1.44 ± 0.87 ng/µg of total protein for wild type, (Arg169 right-arrow Glu), (1-382), and (1-393) forms, respectively). Apparently, faster desensitization in the presence of beta ARK2 reflects stronger binding of the mutants to phosphorylated receptor (Figs. 1, 2). Because the peak agonist-induced beta 2AR and DOR responses were not significantly different in oocytes expressing constitutively active beta -arrestins (compared with oocytes expressing no beta -arrestin or wild type beta -arrestin; data not shown), the mutants do not appear to be prebound to the receptor before agonist application.

Our previous studies demonstrated that the crucial GRK phosphorylation sites are localized on the carboxyl-terminal part of DOR, and that the truncation of the receptor yielding DOR-(1-339) blocked homologous desensitization mediated by beta -arrestin + beta ARK2 (8). We tested whether constitutively active beta -arrestin mutants can rescue the desensitization of DOR-(1-339). Both phosphorylation-independent beta -arrestin mutants induced the desensitization of truncated DOR with virtually the same kinetics as evident for the full-length DOR (Fig. 4). These data suggest that constitutively active beta -arrestins are equally capable of tight binding to (and blocking the signaling of) a receptor without phosphates on the COOH terminus and without the COOH terminus itself. An important implication of this finding is that the major role of the GRK-phosphorylated elements of the receptor is to pull the phosphorylation-sensitive trigger on the arrestin molecule; they do not appear to be required for tight arrestin binding to the receptor per se.


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 4.   Desensitization of truncated DOR-(1-339) by "constitutively active" forms of beta -arrestin. Control oocytes were injected with 0.4 ng of cRNA for DOR-(1-339) and cRNAs for Kir3.1 and Kir3.4 channel subunits, as in Fig. 3. Other oocyte groups were in addition injected with 8 ng of cRNA for beta -arrestin, beta -arrestin-(Arg169 right-arrow Glu), or beta -arrestin-(1-382). DOR-(1-339) responses were elicited by 1 µM DPDPE. Receptor desensitization rates were calculated as described in Fig. 3 and normalized to the response desensitization rate in the oocyte group expressing wild type beta -arrestin. *, p < 0.05, Student's t test, compared with desensitization rate of the group with wild type beta -arrestin.

Thus, the binding of beta -arrestin-(Arg169 right-arrow Glu) and beta -arrestin-(1-382) to unphosphorylated receptor detectable in both in vitro assays (Figs. 1 and 2) translates into the ability of these mutants to induce phosphorylation-independent receptor desensitization in the living cell (Figs. 3 and 4). Taken together, the data corroborate the model of sequential multisite arrestin-receptor interaction (2, 3) and open an enticing prospect of targeted construction of mutant arrestins with different special functional characteristics. Recent studies suggest that arrestin binding targets the receptors for internalization (10, 11), apparently by virtue of the ability of nonvisual arrestins to interact with clathrin (12), which is unaffected by the mutations introduced in this study (data not shown). beta -Arrestin mutants capable of tight phosphorylation-independent binding to the receptor may change the pattern of intracellular receptor trafficking.

Phosphorylation-independent arrestins are likely to prove valuable tools for the experimental manipulation of the efficiency of signaling by different GPCRs. Uncontrolled signaling by various naturally occurring mutant forms of G protein-coupled receptors has been linked to a wide variety of pathological conditions in humans, from stationary night blindness and retinitis pigmentosa (Refs. 13 and 14 and references therein) to Jansen-type metaphyseal chondrodysplasia (15), autosomal dominant hypocalcemia (17, 18), autosomal dominant hyperthyroidism (19, 20), and numerous forms of cancer (reviewed in Ref. 21). Arrestin mutants with an enhanced ability to block this excessive signaling appear promising tools for the gene therapy of these disorders.

    ACKNOWLEDGEMENTS

We thank Dr. J. L. Benovic for purified beta ARK, Dr. J. G. Krupnick for purified rhodopsin kinase, Dr. J. J. Onorato for purified beta 2AR, Dr. J. H. Keen for purified clathrin, and Dr. L. A. Donoso for the arrestin monoclonal antibody F4C1.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grants EY 11500 (to V. V. G.) and DA 04123 (to C. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ These authors contributed equally to this work.

parallel To whom correspondence and requests for materials should be addressed: Ralph & Muriel Roberts Laboratory for Vision Science, Sun Health Research Institute, Sun City, AZ 85372. Tel.: 602-876-5462; Fax: 602-876-6663; E-mail: vgurevich{at}sunhealth.org.

    ABBREVIATIONS

The abbreviations used are: GPCR, G protein-coupled receptor; GRK, G protein-coupled receptor kinase; beta ARK2, beta -adrenergic receptor kinase 2 (GRK3); Kir3, G protein-gated inwardly rectifying K+ channel; beta 2AR, beta 2-adrenergic receptor; P-beta 2AR, phosphorylated beta 2AR; DOR, delta opioid receptor; Rh*, light-activated rhodopsin; P-Rh*, phosphorylated Rh*; HAC, high agonist affinity complex; Ter, stop codon.

    REFERENCES
Top
Abstract
Introduction
References
  1. Freedman, N. J., and Lefkowitz, R. J. (1996) Recent Prog. Horm. Res. 51, 319-353[Medline] [Order article via Infotrieve]
  2. Gurevich, V. V., and Benovic, J. L. (1993) J. Biol. Chem. 268, 11628-11638[Abstract/Free Full Text]
  3. Gurevich, V. V., Dion, S. B., Onorato, J. J., Ptasienski, J., Kim, C. M., Sterne-Marr, R., Hosey, M. M., and Benovic, J. L. (1995) J. Biol. Chem. 270, 720-731[Abstract/Free Full Text]
  4. Granzin, J., Wilden, U., Choe, H.-W., Labahn, J., Krafft, B., and Buldt, G. (1998) Nature 391, 918-921[CrossRef][Medline] [Order article via Infotrieve]
  5. Gurevich, V. V., and Benovic, J. L. (1995) J. Biol. Chem. 270, 6010-6016[Abstract/Free Full Text]
  6. Gurevich, V. V. (1998) J. Biol. Chem. 273, 15501-15506[Abstract/Free Full Text]
  7. Gurevich, V. V., Pals-Rylaarsdam, R., Benovic, J. L., Hosey, M. M., and Onorato, J. J. (1997) J. Biol. Chem. 272, 28849-28852[Abstract/Free Full Text]
  8. Kovoor, A., Nappey, V., Kieffer, B. L., and Chavkin, C. (1997) J. Biol. Chem. 272, 27605-27611[Abstract/Free Full Text]
  9. Kovoor, A., Celver, J. P., Wu, A., and Chavkin, C. (1998) Mol. Pharmacol. 54, 704-711[Abstract/Free Full Text]
  10. Barak, L. S., Ferguson, S. S. G., Zhang, J., and Caron, M. G. (1997) J. Biol. Chem. 272, 27497-27500[Abstract/Free Full Text]
  11. Ferguson, S. S. G., Downey, W. E., III, Colapietro, A.-M., Barak, L. S., Menard, L., and Caron, M. G. (1996) Science 271, 363-366[Abstract]
  12. Goodman, O. B., Jr., Krupnick, J. G., Santini, F., Gurevich, V. V., Penn, R. B., Gagnon, A. W., Keen, J. H., and Benovic, J. L. (1996) Nature 383, 447-450[CrossRef][Medline] [Order article via Infotrieve]
  13. Rao, V. R., Cohen, G. B., and Oprian, D. D. (1994) Nature 367, 639-642[CrossRef][Medline] [Order article via Infotrieve]
  14. Chen, J., Makino, C. L., Peachey, N. S., Baylor, D. A., and Simon, M. I. (1995) Science 267, 374-377[Medline] [Order article via Infotrieve]
  15. Schipani, E., Kruse, K., and Juppner, H. (1995) Science 268, 98-100[Medline] [Order article via Infotrieve]
  16. Gray-Keller, M. P., Detwiler, P. B., Benovic, J. L., and Gurevich, V. V. (1997) Biochemistry 36, 7058-7063[CrossRef][Medline] [Order article via Infotrieve]
  17. Bai, M., Quinn, S., Trivedi, S., Kifor, O., Pearce, S., Pollak, M., Krapcho, K., Hebert, S., and Brown, E. M. (1996) J. Biol. Chem. 271, 19537-19545[Abstract/Free Full Text]
  18. Pearce, S., Trump, D., Wooding, C., Besser, G., Chew, S., Heath, D., Hughes, I., and Thakker, R. (1995) J. Clin. Invest. 52, 736-740
  19. Duprez, L., Parma, J., Van Sande, J., Allgeier, A., Leclere, J., Schvartz, C., Delisle, M. J., Decoulx, M., Orgiazzi, J., Dumont, J. E., and Vassart, G. (1994) Nat. Genet. 7, 396-401[Medline] [Order article via Infotrieve]
  20. Paschke, R., and Ludgate, M. (1997) N. Engl. J. Med. 337, 1675-1681[Free Full Text]
  21. Gutkind, J. S. (1998) J. Biol. Chem. 273, 1839-1842[Free Full Text]
  22. Donoso, L. A., Gregerson, D. S., Smith, L., Robertson, S., Knospe, V., Vrabec, T., and Kalsow, C. M. (1990) Curr. Eye Res. 9, 343-355[Medline] [Order article via Infotrieve]


Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.