©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Rhodopsin Kinase Autophosphorylation
CHARACTERIZATION OF SITE-SPECIFIC MUTATIONS (*)

Krzysztof Palczewski (1) (2)(§), Hiroshi Ohguro (1), Richard T. Premont (3), James Inglese (3)

From the (1)Departments of Ophthalmology and (2)Pharmacology, School of Medicine, University of Washington, Seattle, Washington 98195 and the (3)Departments of Medicine (Cardiology) and Biochemistry, Howard Hughes Medical Institute, Duke University, Durham, North Carolina 27710

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Upon illumination, rhodopsin kinase (RK) phosphorylates the visual pigment rhodopsin, which is thought to partially terminate the biochemical events that follow photon absorption. RK enzymology was explored by mutagenesis of the residues Ser, Thr (major autophosphorylation sites), and Lys (a distal residue). We found the following to be true. (i) Double mutations at residues Ser and Thr to Ala or Asp decrease autophosphorylation to substoichiometrical levels, while single mutations at either residue independently reduce autophosphorylation by half. (ii) Phosphorylation of residue Ser influences the affinity of RK for heparin-Sepharose only moderately, whereas Thr and Lys are important for this interaction. RK K491A does not phosphorylate acidic peptides, suggesting that this residue participates in substrate binding. (iii) Mutations in the autophosphorylation region affect the K for ATP, suggesting that this region is involved in binding of ATP to the catalytic site. (iv) RK mutants S488A or S488D and RK S488A and T489A have an increased ability to phosphorylate Rho in the dark. (v) Mutations at the autophosphorylation region change the initial site of phosphorylation on photolyzed rhodopsin (Rho*), implying that this region may regulate selectivity of the site of phosphorylation.


INTRODUCTION

Rhodopsin kinase (RK)()specifically phosphorylates photoactivated rhodopsin (Rho*) in retinal photoreceptor cells. The phosphorylation of Rho* leads to inhibition of G protein binding (G, transducin) through increased binding of the regulatory protein arrestin (Wilden et al., 1986). Bound arrestin uncouples G from further activation by Rho* and thus terminates the activation pathway of phototransduction (Wilden et al., 1986; Bennett and Sitaramayya, 1988). Arrestin binds to Rho* only when one or two of the several sites at the C terminus of Rho* have been phosphorylated (Ohguro et al., 1994). RK is therefore an important component regulating the level of activation of the G pool on a disc membrane (reviewed by Lagnado and Baylor(1992)). Rho, G, RK, and arrestin are functional archetypes for related G protein-coupled transduction systems that involve different types of receptors, G proteins, G protein-coupled receptor kinases (GRKs), and arrestin isoforms (reviewed by Inglese et al.(1993) and Palczewski and Benovic(1991)).

RK is activated by Rho* and phosphorylates multiple sites on the C terminus of Rho*. Neither opsin nor Rho is a substrate for RK, although the sites of phosphorylation on Rho are accessible. Thus, the binding of RK to the cytosolic surface of Rho* (but not Rho) is required for the subsequent phosphorylation of the C terminus of Rho* (Fowles et al., 1988; Palczewski et al., 1991).

Analysis of the primary sequence reveals that the kinase catalytic domain is located in the midregion of the RK sequence (Lorenz et al., 1991), which has an overall structure related to the -adrenergic receptor kinase (Inglese et al., 1993). The N terminus of RK contains a domain implicated in the recognition of Rho* (Palczewski et al., 1993). RK is post-translationally processed at the C terminus by farnesylation, proteolysis, and -carboxyl methylation (Inglese et al., 1992a), modifications that appear to be important for the light-dependent association of the kinase with Rho* (Inglese et al., 1992b). Additionally, RK is modified by multiple intramolecular phosphorylations at the C and N termini of the enzyme (Lee et al., 1982; Kelleher and Johnson, 1990; Palczewski et al., 1992). Recently, the sites of autophosphorylation in the primary structure of RK were identified as Ser (minor) near the N terminus, and Ser and Thr (major) near the C terminus (Palczewski et al., 1992). Autophosphorylation changes the affinity of RK for different forms of Rho*, with the weakest interaction occurring between phosphorylated RK and phosphorylated Rho*. It was proposed that autophosphorylation of RK facilitates its dissociation from Rho* (Buczyet al., 1991) by changes in the rate of dissociation (k) from phosphorylated Rho* (Pulvermüller et al., 1993).

The goal of the present study was to explore the mechanism of RK regulation by autophosphorylation. Several mutations in the major autophosphorylation sites allowed us to identify changes produced by phosphorylation of these residues with regard to the enzymatic mechanism of RK action.


MATERIALS AND METHODS

Proteins and Reagents

Several forms of Rho were prepared from ROS according to published procedures as follows: urea washed Rho (Shichi and Somers, 1978); phosphorylated Rho by regeneration of phosphorylated opsin with 11-cis-retinal (Palczewski et al., 1989; Hofmann et al., 1992); and G-Rho lacking the C-terminal 19 amino acids by digestion with endoproteinase Asp-N (Palczewski et al., 1991). The catalytic subunit of protein phosphatase 2A (PrP 2A) was purified from rabbit skeletal muscle (Shenolikar and Ingebritsen, 1984) or was a generous gift from Ann A. DePaoli-Roach (Indiana University, Indianapolis). GRKs (2, 3, and 5) were prepared from Sf9 cells using a baculovirus expression system as recently described (Söhlemann et al., 1993; Premont et al., 1994). G was purified from bovine brain using the method of Casey et al.(1989).

Peptide C (DDEASTTVSKTETSQVARRR) and an acidic peptide (RRRDDEEEEESAAA) were synthesized using a fully automated batch instrument (430A, Applied Biosystems, Inc., Foster City, CA) using Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry on 0.1 mmol scale. After deprotection, the peptide was purified by preparative reverse phase HPLC (>95% pure).

Mutagenesis

Site mutations were prepared using standard polymerase chain reaction cassette mutagenesis procedure (Innis and Gelfand, 1990) employing the NcoI and EcoRI sites of RK in the pBlueScript II vector (Stratagene). Following verification of the sequence by dideoxynucleotide sequencing, the RK coding region was subcloned into the eukaryotic expression vector pCMV5 (Andersson et al., 1989).

Purification of RK on a Heparin-Sepharose Column

Bovine RK was purified on a heparin-Sepharose column as described elsewhere (Palczewski, 1993). Bovine RK or mutants were expressed in COS-7 cells transfected with the appropriate plasmid constructs using the diethylaminoethyl dextran method (Inglese et al., 1992a, 1992b). RK or mutants from COS cells were purified as follows. Cells from two 150-mm dishes were harvested by scraping with 75 mM Tris/HCl buffer, pH 7.5, containing 1 mM MgCl, 1 mM dithiothreitol, 20% adonitol, 10 µg/ml leupeptin, and 20 µg/ml aprotinin. The cell suspension was diluted 1:1 with 10 mM BTP buffer, pH 7.5, containing 0.4% Tween 80, homogenized (Tenbroeck Tissue Grinder, Wheaton), and centrifuged at 125,000 g for 7 min (Beckman Optima TLX). The supernatant containing RK was treated with PrP 2A (0.2 µg) or ATP (0.4 mM) for 1 h at room temperature and loaded onto a heparin-Sepharose column (5 50 mm, Pharmacia Biotech Inc.) that had been equilibrated with 10 mM BTP buffer, pH 7.5, containing 0.3% Tween 80. RK was eluted with a NaCl gradient (0-750 mM) in BTP buffer, pH 7.5, containing 0.3% Tween 80 at a flow rate of 30 ml/h using a quaternary HPLC pump system (Hewlett-Packard, model 1050). One-ml fractions were collected, and a protein elution profile was monitored by absorption at 280 nm. Aliquots from all fractions were tested for the presence of RK activity.

Assay for RK Activity

RK activity was measured using [-P]ATP (100-500 cpm/pmol, DuPont NEN) and urea-washed ROS membranes in 20 mM BTP buffer, pH 7.5, at 30 °C (Palczewski, 1993). RK concentration was determined by direct ELISA using recently described anti-RK antibodies (Palczewski et al., 1993) and a known amount of purified RK as a standard.

Identification of the Initial Phosphorylation Sites in Rhodopsin Phosphorylated by GRKs

The initial sites of phosphorylation (monophosphorylated) species of Rho* by mutants of RK and GRKs were evaluated using urea-washed ROS as described for WT RK (Ohguro et al., 1994).


RESULTS AND DISCUSSION

Mutagenesis, Expression, and Purification of RK

Mutations in the RK autophosphorylation domain were as follows. 1) Ala was substituted for Ser and Thr to confirm the sites of autophosphorylation in RK and to determine the contribution of autophosphorylation to RK activity. In addition, we examined the mutation of Lys, distal to the RK autophosphorylation region (see Fig. 1), to determine the influence of this residue on the enzymatic properties of RK. Ala has been used in scanning mutagenesis techniques to remove side chain atoms beyond the -carbon while allowing minimum perturbations in secondary structure and tertiary conformation (Cunningham and Wells, 1989; Gibbs and Zoller, 1991). 2) Asp was employed to mimic the phosphorylated state of the kinase (see Kurland et al. (1992)).


Figure 1: Illustration of RK mutants and GRK family alignment of autophosphorylation region. A, mutations of the autophosphorylation sites examined in this study. B, alignment of the autophosphorylation regions of GRKs 1-6. The dendrogram on the left displays the relative percent homology between the kinases (based on the catalytic domains); this homology is mirrored in the autophosphorylation region of the respective kinases. The boldfaceletters are identical residues among all GRKs, and the underline indicates experimentally determined autophosphorylation sites (Palczewski et al., 1992; Premont et al., 1994). The GRK designation is followed by the common name.



RK expressed in COS cells was purified on DEAE-Sepharose (Palczewski et al., 1988), heparin-Sepharose (Buczyet al., 1991), or a combination of both and yielded 5-25 µg of RK (from two 150-mm plates), which constituted 10-30% of the total protein obtained from these steps. The enzyme was identified by immunoblotting and ELISA using an RK-specific antibody (Palczewski et al., 1993). The RK activity correlated well with the relative amount of RK protein as determined from the ELISA and was specifically inhibited by the RK antibody (data not shown).

Effects of Mutations at the Autophosphorylation Region of RK on Its Interaction with Heparin-Sepharose

Autophosphorylation of RK significantly changed its affinity for heparin (Fig. 2). Using a heparin-Sepharose column, the fully autophosphorylated form of RK was eluted at 90 mM NaCl (fractions 6-7), partially phosphorylated RK was eluted at 200 mM NaCl (fractions 8-9), and dephosphorylated RK was eluted at 290 mM NaCl (fractions 10-12) (Fig. 2A) (Buczyet al., 1991). RK extracts from either ROS, retina (Buczyet al., 1991), or COS cells (data not shown) have similar amounts of unphosphorylated and phosphorylated forms of the enzyme. Thus, RK extracts were dephosphorylated with PrP 2A, and the phosphatase, which binds weakly to heparin (Fowles et al., 1989), was removed during the heparin-Sepharose chromatography step. Employing these steps, dephosphorylated RK was separated from phosphorylated forms before experiments related to the autophosphorylation reaction were performed. This procedure also allowed us to eliminate all interfering phosphorylated proteins and protein kinases present in COS cell extracts. In fact, no protein kinase activity was detected with generic substrates such as casein, histone H2, and Kemptide, a peptide substrate for cyclic-AMP-dependent protein kinase (data not shown). As shown in Fig. 2A, an extract of COS cells that had been transfected with a carrier DNA did not yield any significant phosphorylation of Rho*. All mutants were stable for at least 1 h at 30 °C or 2 days when stored on ice.


Figure 2: Chromatography of RK on a heparin-Sepharose column. The extract from COS cell transfected with RK plasmid was loaded on a heparin-Sepharose column (5 50 mm, Pharmacia), and RK was eluted with a NaCl gradient (0-750 mM) in BTP buffer, pH 7.5, containing 0.3% Tween 80. Aliquots from all fractions were tested for the presence of RK activity. A, RK extract that was preincubated with ATP (opencircles), RK extract that was preincubated with PrP 2A (closedcircles), or COS extract from cells transfected with empty plasmid (opentriangles); B, RK S488A pretreated with ATP (opencircles) or PrP 2A (closedcircles); C, RK S488D,T489D (closedcircles) and RK S488D (opentriangles) pretreated with PrP 2A; D, RK T489A treated with ATP (opencircles) or PrP 2A (closedcircles); E, RK S488A,T489A pretreated with ATP (opencircles) or PrP 2A (closedcircles); F, RK K491A pretreated with ATP (opencircles) or PrP 2A (closedcircles). Dottedlines in all panels corresponded to elution positions of phosphorylated and dephosphorylated WT RK.



The elution profiles of the autophosphorylated or dephosphorylated forms of RK S488A from heparin-Sepharose were shifted by one fraction toward higher salt as compared with the native enzyme. Dephosphorylation of RK S488A by PrP 2A, using conditions that dephosphorylated WT RK, led to only partial dephosphorylation (Fig. 2B), suggesting that RK S488A was a poor substrate for PrP 2A compared with WT RK. Dephosphorylated RK S488D was eluted from the heparin-Sepharose column similarly to WT RK, while the double mutant RK S488D T489D had lower affinity for heparin (Fig. 2C). The elution profile of RK T489A from heparin-Sepharose was significantly different (Fig. 2D). The phosphorylated and dephosphorylated forms of this mutant were eluted close together in a position typical for dephosphorylated WT RK. RK S488A and T489A (Fig. 2E) was insensitive to ATP or PrP 2A treatment. These results suggest that phosphorylation of residue Ser influences the affinity of RK for heparin-Sepharose only moderately, whereas Thr residue was more critical for this interaction. Because RK K491A had lower affinity for heparin (Fig. 2F), residue Lys may comprise part of the RK heparin binding site. This effect was reflected in the lower IC of heparin for WT RK (0.3 mM) versus RK K491A (>10 mM) (data not shown).

Effects of Mutations at the Autophosphorylation Region of RK on the Stoichiometry and Rate of Autophosphorylation

Autophosphorylation of WT RK and RK K491A (data not shown) had similar stoichiometries and rates, while single mutations at residues 488 or 489 to Ala or Asp yielded a kinase with autophosphorylation stoichiometries reduced by half, as shown, for example, for RK S488D (Fig. 3). Double mutations at residues Ser and Thr to Ala or Asp almost eliminated autophosphorylation (Fig. 3). Each of these residues, Ser or Thr, could be phosphorylated independent of the other, indicating that there was no hierarchical phosphorylation. The RK/GRK2 chimera did not undergo phosphorylation (data not shown). The overall stoichiometry of autophosphorylation was lower than anticipated, which may be explained by a fraction of the RK being denatured or the RK concentration being consistently overestimated.


Figure 3: Time course and stoichiometry of RK autophosphorylation. A, autoradiogram of RK and RK S488D,T489D autophosphorylation. B, time course and stoichiometry of RK autophosphorylation. From the top: WT RK (closedcircles); RK S488D (opentriangles); and RK S488D,T489D (opencircles). Known amounts of purified, dephosphorylated RK (1-5 µg) were incubated with 10 µM [-P]ATP (3,000-6,000 cpm/pmol) in BTP buffer containing 1 mM MgCl, 100 mM NaCl at 30 °C. At selected time points, aliquots of the reaction mixture were removed, mixed with an equal volume of 10% SDS, and analyzed by SDS-polyacrylamide gel electrophoresis (Laemmli, 1970). Radioactive bands were visualized by autoradiography, cut out, dissolved in 30% HO, and mixed with scintillation counting solution. Radioactivity was measured with a scintillation counter.



Kinetic Parameters of RK Mutants

The Kfor Rho* (2-4 µM) was similar for WT RK and all mutants with changes in the autophosphorylation region (). RK S488A and RK S488A,T489A had a 20-fold higher K for ATP, while RK S488D and RK S488D,T489D and the chimeric mutant had a K similar to WT RK, suggesting that Asp can be utilized for the hydrogen bond network with the phosphates of ATP. While Thr mutants had no effect on Kfor ATP, double mutant RK S488A,T489A had the highest V, suggesting that Ser and Thr residues may also influence the ATP phosphotransfer.

In a centrifugation assay (Buczyet al., 1991), dephosphorylated WT RK bound to phosphorylated Rho*. In our experimental conditions, 70% of RK is associated with membranes, while we did not detect a stable complex between RK S488D,T489D and P-Rho* (data not shown), supporting an idea that autophosphorylation may influence the rate of RK dissociation from P-Rho*.

Phosphorylation of a Model Peptide

Following endopeptidase Asp-N cleavage, all phosphorylation sites in the Rho molecule are removed; however, the resulting truncated Rho* (G-Rho*) can still bind RK and stimulate phosphorylation of an exogenous acidic peptide (Palczewski et al., 1991). Although the mechanism of peptide phosphorylation by RK is probably different from that involving receptor phosphorylation as reflected by large differences in V/K (Palczewski et al., 1989), the assay of peptide phosphorylation in the presence of G-Rho* might reveal differences between mutated RKs (). Phosphorylation of the model peptide by the majority of RK mutants occurs at similar rates (4 ÷ 5 nmol of P/(min mg of RK)) and was not appreciably enhanced by G-Rho*, in contrast to the 4-fold enhancement seen with WT RK. RK K491A, which interacted poorly with heparin, did not phosphorylate acidic peptides, suggesting that these residues participate in binding or catalysis of peptide phosphorylation. These results indicated that the ability of Rho* to stimulate peptide phosphorylation is altered when a mutation at the autophosphorylation domain is present.

SerMutants of RK Are Active in the Dark

RK binds to Rho*, presumably to the metarhodopsin I and II conformers (Pulvermüller et al., 1993; Paulsen and Bentrop, 1984). However, several lines of evidence suggest that the C-terminal region of Rho, the site of multiple phosphorylations catalyzed by RK, is already exposed and available for interaction with enzymes before Rho assumes the activated Rho* conformation (discussed in Palczewski et al.(1991)). Here we found that a mutant with Ser converted to Ala has 7 ± 1% of activity toward Rho (in dark) compared with phosphorylation of Rho* (in light). Mutant RK S488D and double mutant RK S488A,T489A had the capability to phosphorylate Rho in the dark 5 ± 1 and 11 ± 1%, respectively. WT RK and other mutants were inactive in phosphorylating Rho in the dark. Also, RK S488A, RK S488D, and RK S488A,T489A had higher affinity for Rho than WT RK. In the centrifugation assay, 20-30% of the RK mutants were associated with the membranes, compared with less than 5% for the WT RK (data not shown).

Sites of Rho* Phosphorylation by RK Mutants and GRKs

The initial sites of Rho* phosphorylation were studied employing conditions recently described (Ohguro et al., 1994) with some modifications. Urea-washed bovine ROS were phosphorylated by RK or its mutants in conditions that favored a phosphorylation stoichiometry of 1 mol of P/mol of Rho*. The C-terminal 19-residue peptide of Rho, containing all sites phosphorylated by RK, was analyzed by proteolytic subdigestion of P-labeled peptides on HPLC. Standard phosphopeptide sequences were verified by mass spectroscopy as described recently (Ohguro et al., 1994). Similar to the phosphorylation by RK isolated from ROS, the COS-expressed WT enzyme phosphorylated mainly Ser and to a lesser extent Ser on Rho* (Fig. 4). Mutations of RK Ser or Thr to Ala made these mutants almost completely selective for Rho* Ser. Mutation of Ser to Asp changed this specificity back to that of WT RK, as the primary sites of phosphorylation were observed to be at Rho* Ser and Rho* Ser. To explore this important observation further, three additional GRKs and a RK/GRK2 chimera were tested in this assay. Initial sites of phosphorylation by GRK2, GRK3, and RK/GRK2, which have the highly charged amino acid sequence DEED in the autophosphorylation region (Fig. 1), were different from that obtained for RK S488D,T489D mutants, suggesting that other regions than the autophosphorylation domain also are involved in the substrate specificity. G subunits, which are known to accelerate receptor phosphorylation by GRK2 and -3 (reviewed by Inglese et al. (1993)), had no effect on the initial sites of phosphorylation, suggesting that the action of these subunits is to bring these GRKs to membranes rather than to change the mechanism of phosphorylation. GRK5, which has similar sequence to RK and undergoes autophosphorylation in sites equivalent to these RK sites (Premont et al., 1994), yielded similar phosphorylation to WT RK. These results suggest that the autophosphorylation region may influence the stability of the activated form of RK, thereby leading to modification of the receptor at specific sites.


Figure 4: Initial sites of phosphorylation of Rho* by WT RK, RK mutants, and other GRKs. The bargraph shows the relative degree of phosphorylation for individual Ser residues in the C terminus of Rho* by each kinase studied for monophospho-Rho*. The C-terminal sequence of Rho* in which these initial sites of phosphorylation occur is shown below. The differences between experiments were below 5%.



Autophosphorylation of RK exhibits clearly distinct features from that of GRK5 (Premont et al., 1994; Kunapuli et al., 1994). (i) In contrast to GRK5 the autophosphorylation of RK is not influenced by ROS membranes or Rho* (Kelleher and Johnson, 1990). (ii) Differently autophosphorylated RK and autophosphorylation mutants display strong effects on the affinity to heparin, whereas a GRK5 mutant was not affected by heparin (Kunapuli et al., 1994). Interestingly, heparin only modestly inhibits RK (K0.3 mM) and is a very potent inhibitor of GRK5 (150 nM). Thus, it is conceivable that both enzymes utilize different mechanisms of heparin binding. (iii) Mutants in the autophosphorylation domain have similar kinetic parameters for RK, while GRK5 mutants phosphorylate poorly agonist-occupied receptors, possibly due to a decrease in K for ATP. The functional consequences of autophosphorylation of GRK5 and GRK6 remain to be established.

  
Table: Effects of mutations in the autophosphorylation region of RK on the kinetics of Rho* and peptide phosphorylation

The K for Rho* was determined by varying the Rho* concentration from 0.2 to 20 µM at an ATP concentration of 100 µM. The reaction time (with illumination) was 10 min. The K for ATP and V were determined by varying the ATP concentration from 0.2 to 20 times the estimated K at a Rho* concentration of 20 µM. The reaction time (with illumination) was 10 min. The amount of RK used in the reactions was chosen so that no more than 5% of Rho* was phosphorylated and less than 1% of ATP was used during this assay. The K values were calculated by Lineweaver-Burk analysis as described by Segel (1975). Rho concentration was determined spectrophotometrically at 498 nm assuming a molar absorption coefficient of 40,600 (Wald and Brown, 1953) and a molecular mass of 40 kDa. ATP concentration was determined spectrophotometrically.



FOOTNOTES

*
This research was supported by United States Public Health Service Grants EY08061 (to K. P.) and HL16037 (to R. J. Lefkowitz) and by an award to the Department of Ophthalmology at the University of Washington from Research to Prevent Blindness Inc. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of a Jules and Doris Stein Research to Prevent Blindness Professorship. To whom correspondence should be addressed: Dept. of Ophthalmology, RJ-10, School of Medicine, University of Washington, Seattle, WA 98195. Tel.: 206-543-9074; Fax: 206-543-4414.

The abbreviations used are: RK, rhodopsin kinase; BTP, 1,3-bis[tris(hydroxymethyl)methylamino]propane; G, G-protein of the rod cell transducin; Rho*, photolyzed rhodopsin; WT RK, wild-type rhodopsin kinase; ROS, rod outer segment(s); GRK, G protein-coupled receptor kinase; PrP, protein phosphatase; HPLC, high pressure liquid chromatography; ELISA, enzyme-linked immunosorbent assay.


ACKNOWLEDGEMENTS

We especially thank Dr. Robert J. Lefkowitz (Duke University) in whose laboratory numerous experiments reported here were performed. We thank J. P. Van Hooser, H. Kendall, C. Brown, and G. Irons for excellent technical assistance. We thank Dr. Janina Buczyfor help with RK assays during the course of this work, Dr. Ann A. DePaoli-Roach for the generous gift of PrP 2A, and Dr. Ann Milam for comments on the manuscript.


REFERENCES
  1. Andersson, S., Davis, D. L., Dahlback, H., Jornvall, H., and Russell, D. W.(1989) J. Biol. Chem.264, 8222-8229 [Abstract/Free Full Text]
  2. Bennett, N., and Sitaramayya, A.(1988) Biochemistry27, 1710-1715 [Medline] [Order article via Infotrieve]
  3. Buczy, J., Gutmann, C., and Palczewski, K.(1991) Proc. Natl. Acad. Sci. U. S. A.88, 2568-2572 [Abstract]
  4. Casey, P. J., Graziano, M. P., and Gilman, A. G.(1989) Biochemistry28, 611-616 [Medline] [Order article via Infotrieve]
  5. Cunningham, B. C., and Wells, J. A.(1989) Science244, 1081-1085 [Medline] [Order article via Infotrieve]
  6. Fowles, C., Sharma, R., and Akhtar, M.(1988) FEBS Lett.238, 56-60 [CrossRef]
  7. Fowles, C., Akhtar, M., and Cohen, P.(1989) Biochemistry28, 9385-9391 [Medline] [Order article via Infotrieve]
  8. Gibbs, C. S., and Zoller, M. J.(1991) J. Biol. Chem.266, 8923-8931 [Abstract/Free Full Text]
  9. Hofmann, K. P., Pulvermüller, A., Buczy, J., Van Hooser, P., and Palczewski, K.(1992) J. Biol. Chem.267, 15701-15706 [Abstract/Free Full Text]
  10. Inglese, J., Glickman, J. F., Lorenz, W., Caron, M. G., and Lefkowitz, R. J. (1992a) J. Biol. Chem.267, 1422-1425 [Abstract/Free Full Text]
  11. Inglese, J., Koch, W. J., Caron, M. G., and Lefkowitz, R. J. (1992b) Nature359, 147-150 [CrossRef][Medline] [Order article via Infotrieve]
  12. Inglese, J., Freedman, N. J., Koch, W. J., and Lefkowitz, R. J.(1993) J. Biol. Chem.268, 23735-23738 [Free Full Text]
  13. Innis, M. A., and Gelfand, D. H.(1990) in PCR Protocols: A Guide to Methods and Application (Innis, M. A., ed) pp. 1-482, Academic Press, Inc., San Diego, CA
  14. Kelleher, D. J., and Johnson, G. L.(1990) J. Biol. Chem.265, 2632-2639 [Abstract/Free Full Text]
  15. Kunapuli, P., Gurevich, V. V., and Benovic, J. L.(1994) J. Biol. Chem.269, 10209-10212 [Abstract/Free Full Text]
  16. Kurland, I. J., el-Maghrabi, M. R., Correia, J. J., and Pilkis, S. J. (1992) J. Biol. Chem.267, 4416-4423 [Abstract/Free Full Text]
  17. Laemmli, U. K.(1970) Nature227, 680-685 [Medline] [Order article via Infotrieve]
  18. Lagnado, L., and Baylor, D.(1992) Neuron8, 995-1002 [Medline] [Order article via Infotrieve]
  19. Lee, R. H., Brown, B. M., and Lolley, R. N.(1982) Biochemistry21, 3303-3307 [Medline] [Order article via Infotrieve]
  20. Lorenz, W., Inglese, J., Palczewski, K., Onorato, J. J., Caron, M. G., and Lefkowitz, R. J.(1991) Proc, Natl. Acad. Sci. U. S. A.88, 8715-8719 [Abstract]
  21. Ohguro, H., Johnson, R. S., Ericsson, L. H., Walsh, K. A., and Palczewski, K.(1994) Biochemistry33, 1023-1028 [Medline] [Order article via Infotrieve]
  22. Palczewski, K.(1993) Methods Neurosci.15, 217-225
  23. Palczewski, K., and Benovic, J. L.(1991) Trends Biol. Sci.16, 387-391
  24. Palczewski, K., McDowell, J. H., and Hargrave, P. A.(1988) J. Biol. Chem.263, 14067-14073 [Abstract/Free Full Text]
  25. Palczewski, K., Arendt, A., McDowell, J. H., and Hargrave, P. A.(1989) Biochemistry28, 8764-8770 [Medline] [Order article via Infotrieve]
  26. Palczewski, K., Buczy, J., Kaplan, M. W., Polans, A. S., and Crabb, J. W.(1991) J. Biol. Chem.266, 12949-12955 [Abstract/Free Full Text]
  27. Palczewski, K., Buczy, J., Van Hooser, P. J., Carr, S. A., Huddleston, M. J., and Crabb, J. W.(1992) J. Biol. Chem.267, 18991-18998 [Abstract/Free Full Text]
  28. Palczewski, K., Buczy, J., Lebioda, L., Crabb, J. W., and Polans, A. S.(1993) J. Biol. Chem.268, 6004-6013 [Abstract/Free Full Text]
  29. Paulsen, R., and Bentrop, J.(1984) Nature302, 417-419
  30. Premont, R. T., Koch, W. J., Inglese, J., and Lefkowitz, R. J.(1994) J. Biol. Chem.269, 6832-6841 [Abstract/Free Full Text]
  31. Pulvermüller, A., Palczewski, K., and Hofmann, K. P.(1993) Biochemistry32, 14082-14088 [Medline] [Order article via Infotrieve]
  32. Segel, I. H.(1975) Enzyme Kinetics, Wiley-Interscience, John Wiley & Sons, Inc., New York
  33. Shenolikar, S., and Ingebritsen, T. S.(1984) Methods Enzymol.107, 102-129 [Medline] [Order article via Infotrieve]
  34. Shichi, H., and Somers, R. L.(1978) J. Biol. Chem.253, 7040-7046 [Medline] [Order article via Infotrieve]
  35. Söhlemann, P., Hekman, M., Buchen, C., Elice, J. S., and Lohse, M. J.(1993) FEBS Lett.324, 59-62 [CrossRef][Medline] [Order article via Infotrieve]
  36. Wald, G., and Brown, P.(1953) J. Gen. Physiol.37, 189-200 [Free Full Text]
  37. Wilden, U., Hall, S. W., and Kühn, H.(1986) Proc. Natl. Acad. Sci. U. S. A.83, 1174-1178 [Abstract]

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