November 2003
Volume 44, Issue 11
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Biochemistry and Molecular Biology  |   November 2003
Cone Photoreceptor βγ-Transducin: Posttranslational Modification and Interaction with Phosducin
Author Affiliations
  • Fayu Chen
    From the Jules Stein Eye Institute, UCLA School of Medicine, Los Angeles, California; the
  • Pei-San Ng
    Molecular Neurology Laboratory, VA Greater Los Angeles Healthcare System at Sepulveda, Sepulveda, California; the
  • Kym F. Faull
    Pasaraw Mass Spectrometry Laboratory, Department of Chemistry, Biochemistry, Psychiatry and Behavioral Science, and The Neuropsychiatry Institute, UCLA, Los Angeles, California.
  • Rehwa H. Lee
    From the Jules Stein Eye Institute, UCLA School of Medicine, Los Angeles, California; the
    Molecular Neurology Laboratory, VA Greater Los Angeles Healthcare System at Sepulveda, Sepulveda, California; the
Investigative Ophthalmology & Visual Science November 2003, Vol.44, 4622-4629. doi:10.1167/iovs.03-0420
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      Fayu Chen, Pei-San Ng, Kym F. Faull, Rehwa H. Lee; Cone Photoreceptor βγ-Transducin: Posttranslational Modification and Interaction with Phosducin. Invest. Ophthalmol. Vis. Sci. 2003;44(11):4622-4629. doi: 10.1167/iovs.03-0420.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. To characterize the structure of cone βγ-transducin (Tβ3γ8) and its interaction with phosducin (pdc).

methods. The Tγ8 subunit of Tβ3γ8 was isolated by column chromatography for peptide mapping with mass spectrometry. Tβ3γ8 was compared with rod βγ-transducin (Tβ1γ1) in terms of the electrophoretic mobility, pdc binding affinity, and the effects of phosphorylation and methylation, and then the correlation to the crystal structures and functional domains of Tβ1γ1 was determined.

results. The mature Tγ8 is a 65-amino-acid peptide encoded by the Gγ8 gene with an acetylated and a farnesylated–methylated N- and C-terminus, respectively. Purified Tβ3γ8 is similar to Tβ1γ1 in that (1) both are heterogeneous, containing methylated and demethylated Tγ subunits; (2) each demethylated dimer migrates faster than its methylated counterpart during native gel electrophoresis, and the methylation-associated mobility differential is masked by pdc binding; and (3) both dimers bind pdc with the same affinity, and the affinity is reduced threefold by PKA phosphorylation of pdc and twofold by demethylation at the C-terminus of Tγ. Tβ3γ8 differs from Tβ1γ1 in exhibiting lower intrinsic electrophoretic mobility, and the difference is unaffected by either pdc binding or the status of Tγ methylation.

conclusions. Tβ3γ8 is identical with Tβ1γ1 in Tγ isoprenylation, the spatial organization, and the mode of pdc binding, indicating that its interaction with pdc does not play an important role in the specialization of cones. Changes in Tβγ characteristics by Tγ methylation reveal conformational changes on a surface domain that is essential for Tβγ functions and support a regulatory role for reversible methylation.

The family of large G proteins plays an essential role in transducing extracellular signals from cell-surface receptors to intracellular effectors. 1 2 Members of this family are heterotrimers composed of Gα, Gβ, and Gγ subunits, with the Gβ and Gγ subunits forming a tightly associated Gβγ dimer and acting as a single functional unit. In rods and cones, the conversion of light signals into neuronal signals (phototransduction) is mediated by the cell-specific G protein, transducin (TαTβγ). 3 4 On activation by light, TαTβγ dissociates and Tα in turn activates a phosphodiesterase (PDE) that reduces the intracellular concentrations of cGMP and ultimately triggers membrane hyperpolarization. Light also triggers a plethora of other reactions that orchestrate the deactivation of phototransduction proteins and the resynthesis of cGMP to ensure timely termination of membrane hyperpolarization and the return of photoreceptors to the dark condition. 5 6  
The βγ-subunit of transducin (Tβγ) regulates several reactions in the light responses, such as promoting the receptor–transducin interaction, 3 4 regulating the transducin–PDE interaction, 7 and regulating the activity of guanylate cyclase, 8 an enzyme that synthesizes cGMP. The normal functions of Tβγ require proper hydrophobic translational modifications at the C-terminus of the Tγ subunit. The nascent Tγ, like other members of the Gγ family, contains a C-terminal CAAX motif that signals three steps of reactions collectively called isoprenylation: (1) the addition of a polyisoprenyl group to the cysteine (C) residue; (2) proteolytic cleavage next to the modified cysteine to remove the AAX residues; and (3) methylation of the carboxyl group of the newly generated C-terminal cysteine. 9 10 Of the three reactions, only the last methylation is reversible and is believed to be a potential step for regulating the activities of isoprenylated proteins. 11 All Gγ isoforms are modified by the geranylgeranyl group, a 20-carbon isoprenoid, 9 10 except that Tγ is modified by a farnesyl group, a 15-carbon isoprenoid. Because the latter isoprenoid is less hydrophobic, it has been suggested that methylation plays a more important regulatory role in Tβγ functions. 11  
The light responses of rods and cones differ in their sensitivity and kinetics. Compared with rods, cones are less sensitive to light, respond and recover more quickly, and adapt to a wider range of illumination. 3 Considerable evidence has accumulated to indicate that the deactivation of phototransduction plays a major role in the rod and cone differences. 5 6 12 13 The primary proteins of the cone phototransduction cascade are isoforms that are distinct from those of rods, whereas many proteins involved in deactivation and recovery are shared. 3 5 6 12 13 Transducins from rods and cones are composed of the Tα1, Tβ1, Tγ1, and the Tα2, Tβ3, Tγ8 subunits. 14 15 16 Mutations in Tα1 17 and Tα2 18 have been found to be associated with visual defects in rods and cones, respectively, but it is not known how these isoforms contribute to the specialization of rod and cone physiology. Because of the scarcity of cones in most mammalian retinas, the biochemistry of cone transducin is not as well characterized as the rod counterpart. A cDNA encoding for cone Tγ8 has been characterized. 16 The predicted amino acid sequence contains the CAAX motif, but the structure of the mature Tγ8 has not been determined. It has been reported that cone transducin 19 and cone PDE 20 are more soluble than their rod counterparts. In view of the role of isoprenylation in membrane targeting, it is of interest to determine whether the C-terminus of Tγ8 is indeed posttranslationally modified in the same manner as Tγ1. 
In mammalian photoreceptors, Tβγ also binds tightly to phosducin (pdc), an abundant phosphoprotein that is phosphorylated by PKA and by Ca2+/calmodulin protein kinase II (CAMKII). 21 22 23 The affinity of pdc for Tβγ is reduced modestly by PKA phosphorylation at one serine residue 24 but drastically after multisite phosphorylation by CAMKII. 23 In the photoreceptor, the levels of phosphorylated pdc are highest in the dark and lowest in the light. 25 In vitro, pdc inhibits the light-activated PDE by binding to Tβγ and inhibiting the receptor–transducin interaction. The inhibition is abolished by phosphorylation. 23 26 27 Pdc also inhibits ubiquitylation of Tβγ and the inhibition is abolished by phosphorylation. 28 It has been reported that transgenic mice expressing pdc with a defective PKA phosphorylation site exhibit reduced sensitivity to light as well as slow and incomplete recovery from exposure to light (Hamasaki DI, et al. IOVS 1996;37:ARVO Abstract 3720). Moreover, these mice show slow retinal degeneration, indicating that normal and reversible phosphorylation of pdc is essential for the viability of photoreceptor cells (Hamasaki DI, et al. IOVS 1995;36:ARVO Abstract 2933). Besides the outer segment, both pdc and Tβγ are found abundantly in the photoreceptor cell body, the synaptic terminal, and the nucleus. 21 29 Clearly, the light- and phosphorylation-regulated interaction between pdc and Tβγ represents a key regulatory step in coordinating light-regulated photoreceptor activities. Our laboratory has recently shown that pdc expressed by bovine cones is identical with rod pdc, and it also colocalizes with Tβγ throughout the cone photoreceptor (Lee RH, et al., unpublished observations, 2003). A comparison between the binding of pdc to the rod and cone Tβγ will provide information to indicate or exclude a role for pdc and Tβγ interaction in the specialized rod and cone physiology. 
We have developed a purification protocol that separates cone Tβγ from the very similar and much more abundant rod Tβγ. The present study was focused on the posttranslational modification of Tγ8, the interaction between pdc and cone Tβγ, and how the interaction is affected by posttranslational modifications on both Tγ8 and pdc. By comparing the characteristics of rod and cone Tβγ against the crystal structures of rod Tβγ and the rod pdc/Tβγ complex, we determined that cone Tβγ has the same conformation and interacts with pdc in the same manner as rod Tβγ. Most interesting, changes in Tβγ characteristics by Tγ methylation reveal conformational changes on a surface domain that is essential for Tβγ functions and support a regulatory role for reversible methylation. In the remainder of this report, Tβγ is used to refer to the general characteristics of βγ-transducin. The rod and cone Tβγ are referred to as Tβ1γ1 and Tβ3γ8, respectively, when the identity of the individual subunit is essential for clarity. 
Methods
Preparation of Cone Tβ3γ8
Pdc/Tβ1γ1, pdc, Tβ1γ1, and a fraction enriched in pdc/Tβ3γ8 were purified from frozen bovine retinas as described. 15 The pdc/Tβ3γ8-enriched fraction was further separated from pdc/Tβ1γ1 by a separation column (CHT10-I; Bio-Rad, Hercules, CA) pre-equilibrated in 10 mM potassium phosphate (pH 6.8) at 25°C and eluted with a 10- to 90-mM phosphate gradient at 1.5 mL/min over a period of 40 minutes, and a peak containing purified pdc/Tβ3γ8 was eluted by 60 mM phosphate. To obtain Tβ3γ8, pdc/Tβ3γ8 was applied to a Sepharose column (1.6 × 15 cm, Q-Sepharose; Amersham Bioscience, Piscataway, NJ) pre-equilibrated at room temperature with 0.3 M Tris-HCl (pH 8.0) and eluted at 3 mL/min with a 400 mL 0.3- to 0.8-M Tris-HCl linear gradient. Tβ3γ8 was eluted as a sharp peak at the beginning of the gradient. 
Reversed-Phase HPLC
The Pdc/Tβ3γ8 or pdc/Tβ1γ1 complex, dissolved in 6 M guanidine HCl, was injected onto a silica based C4 column (BioRad RP-304, 4.6 × 250 mm). The solvent consisted of 0.1% aqueous trifluoroacetic acid (TFA, solvent A) and 95% acetonitrile containing 0.1% TFA (solvent B). The column was pre-equilibrated in 5% B, and the bound proteins were eluted at 1 mL/min by a 60-minute linear gradient from 5% to 100% B. The effluent was monitored by absorbance at 280 nm, and 0.5-mL fractions were collected. Individual fractions were subjected to SDS-PAGE followed by Coomassie brilliant blue (CBB) staining and by Western blot analysis with antisera against the protein of interest. 
Enzymatic Digestion of Tγ8
Tγ8 was dissolved in 50 μL of 50 mM NH4HCO3 (pH 9.5) and incubated with Arg-C or Lys-C (CalBiochem, La Jolla, CA) at room temperature for 16 hours at the substrate-to-enzyme ratio of 100:1. The digestion was stopped by freezing and lyophilization. 
Electrospray Ionization Mass Spectrometry
The masses of intact Tγ8 and its peptide fragments were measured by a triple quadrupole mass spectrometer (API III; Perkin-Elmer Sciex, Thornhill, Ontario, Canada) fitted with an ion spray source. Positive ion protein spectra were produced by injection of the intact proteins or proteolytic peptides dissolved in water/acetonitrile/TFA (95/5/0.1, vol/vol/vol). Data were recorded with the mass spectrometer scanning from m/z 300 to 2200 (step size 0.3 Da, dwell time 1 ms, 6.66 seconds/scan, orifice voltage 90 V for intact proteins or 65 V for tryptic peptides). The average of the spectra contributing to the peak in ion current was computed. Calculation of molecular weights from the series of multiply charged ions found in the spectra was performed on computer (MacSpec software, ver.3.3; Perkin-Elmer Sciex). The theoretical protein or peptide average (chemical) molecular weights were downloaded from http:\\prospector.ucsf.edu. 
Antibodies and Other Biochemical and Analytical Procedures
Antisera against pdc (gertie), Tβ1 (β-636), Tβ3 (β-638), and Tγ8 have been described. 15 16 24 Anti-Tγ1 15 was a kind gift from Bernard Fung (UCLA). The PKA catalytic subunit was purified from rabbit skeletal muscle as described. 21 PKA phosphorylation, SDS-PAGE, and the bandshift binding assay for pdc and Tβγ interaction were performed as described. 24 Native-PAGE 24 was performed at either pH 7.5 or 8.8 and at either 4°C or 22°C, as indicated in text. The separated protein bands were visualized by CBB staining and identified by comigration with standard proteins and by Western blot analysis with appropriate antibodies. The intensities of CBB-stained bands and immunoreactive bands were quantified by densitometric scanning (GS700 densitometer; Bio-Rad). 
Results
Separation of Rod Tγ1 and Cone Tγ8 by RP-HPLC
Pdc/Tβ1γ1, in parallel with pdc/Tβ3γ8, was subjected to reverse-phase (RP)-HPLC. The elution of proteins was monitored by absorbance at 280 nm (Fig. 1) , CBB staining, and Western blot analysis of individual fractions, using antisera against Tγ1, Tγ8, Tβ1, Tβ3, and pdc (results not shown). The elution profiles from both protein complexes were similar and contained multiple absorbance peaks, indicating subunit dissociation. Tγ1 was detected in the fraction corresponding to absorbance peak Gr (33.7 minutes and 36% acetonitrile). Tγ8 was detected in both peak Gc1 and peak Gc2 (30.5–31.5 minutes and approximately 33%–35% acetonitrile). Thus, RP-HPLC is useful for the complete separation of cone Tγ8 from rod Tγ1. The other subunits from both complexes eluted similarly. Pdc was detected in fractions corresponding to the absorbance peak at 38 minutes and 40% acetonitrile. The Tβ1 and Tβ3 subunits, respectively, were detected in multiple fractions eluting between 35 and 37 minutes. The immunoreactive signals for both peptides were much weaker than those of Tγ and pdc, and the respective profiles did not match the sharp UV absorbance peaks detected in the same region. The poor recovery of the Tβ1 and the Tβ3 peptides from the hydrophobic column is consistent with previous observations 30 and their hydrophobic nature. In fact, none of the sharp UV-absorbing peaks could be correlated with any CBB stained bands, suggesting that they are associated with small molecules not fixed on the SDS gels. The nature of the absorbing substance(s) was not investigated further. 
Determination of the Mass of Mature Cone Tγ8
The open reading frame in the cDNA of bovine cone Tγ8 translates into a 69-amino-acid peptide, 16 but the mass and the structure of the mature Tγ8 protein have not been determined. Electrospray ionization mass spectrometry (ESIMS) of Tγ1 (peak Gr) revealed two masses of 8330.1 and 8316.2 Da, (Fig. 1A , inset) which agree with the reported values for the methylated (8329.7 Da) and the demethylated (8315.7 Da) Tγ1. 31 ESIMS of Tγ8 in Peak Gc1 and peak Gc2 also revealed two masses (7529.9 and 7544.1 Da), with the lower mass present predominantly in peak Gc1 and the higher mass in peak Gc2 (Fig. 1B , insets). Based on the total ion current, the lighter and the heavier masses were estimated to compose approximately 15% and 85%, respectively, of the total Tγ8. Together, these indicate that there are two forms of Tγ8 that are separable by RP-HPLC—the heavier and major form being more hydrophobic. The mass of the major Tγ8 is 185 Da lower than the theoretical mass (7728.0 Da) of the deduced Tγ8 sequence, but is 42 Da higher than the theoretical mass, which assumes that Tγ8 undergoes the same posttranslational modifications as Tγ1—namely, isoprenylation with a farnesyl group at the C-terminus and the removal of methionine from the N-terminus of the nascent Tγ1 peptide. 31 Because Tγ8 was not amenable to protein sequencing, 16 the extra 42 Da is interpreted to indicate an acetyl group (42 Da) that blocks the new N-terminus generated by the removal of methionine. The minor Tγ8, being 14 Da lighter and less hydrophobic, is likely the demethylated form of Tγ8 (14 Da for —CH2). 
Mass Peptide Mapping of Cone Tγ8
To confirm the proposed posttranslational modifications, Tγ8 was digested with either Arg-C or Lys-C. The masses of the resultant fragments were mapped to the proposed structure of the mature Tγ8 (Fig. 2) . ESIMS of the Arg-C digest revealed four molecules with observed masses of 7529.9, 4522.4, 4508.2, and 3039.1 Da. The heaviest mass is the same as that of the minor Tγ8, indicating incomplete digestion by Arg-C. The lightest mass matched the theoretical mass (3038.7 Da) of the N-terminally acetylated 1-25 peptide. The remaining pair of masses matched the theoretical masses (4522.0 and 4508.0 Da) for the methylated and demethylated forms, respectively, of the C-terminally farnesylated 26-65 peptide. Likewise, ESIMS of the Lys-C digest identified five fragments that covered 100% of the Tγ8 sequence with the proposed posttranslational modifications. These results conclusively established that the mature Tγ8 is a 65-amino-acid peptide with an acetylated N-terminus and a farnesylated and methylated C-terminus. 
Native-PAGE of Cone Tβ3γ8: The Effects of Methylation and pdc Binding
The migration patterns of pdc/Tβ3γ8 and Tβ3γ8 during native-PAGE at pH 7.5 and 4°C were compared, and the identity of each stained protein band was established by Western blot analysis with antisera against Tβ3, Tγ8, and pdc (Fig. 3A) . The pdc/Tβ3γ3 complex migrated as a single band with an average mobility of 0.41. In contrast, Tβ3γ8, which was judged to be homogeneous by SDS-PAGE and CBB staining of the Tβ3 band, separated into two bands with an average electrophoretic mobility of 0.25 and 0.30. The relative intensity between the slow- and fast-moving bands varied from 10:1 to 1:1, among different Tβ3γ8 samples, with the intensity of the fast-moving band notably higher in the older preparations. Because demethylation at the C-terminus of Tγ8 introduces one additional negative charge, the slow and fast bands may be associated with the methylated and the demethylated forms of Tβ3γ8, respectively. Indeed, comparison of results from four different Tβ3γ8 samples showed that the relative intensity of the fast and slow bands paralleled the relative intensity of peaks Gc1 and Gc2, respectively, in the corresponding RP-HPLC profile (Fig. 1B) . To further ascertain our band assignment, we incubated Tβ3γ8 with immobilized pig liver esterase (iPLE), an enzyme shown to cleave the C-terminus methyl ester bond selectively. 30 As expected, this treatment resulted in the disappearance of the slow band with concomitant increase in the intensity of the fast band (Fig. 3B)
The mobility of the pdc/Tβ3γ8 complex during native-PAGE at pH 7.5 was higher than Tβ3γ8, apparently as the result of binding to the more acidic pdc. The migration of the complex as a single band also suggests that pdc binding masks the heterogeneity in Tγ8 methylation that was detected by both RP-HPLC and mass spectrometric analysis (Figs. 1 2) . To confirm this notion, pdc/Tβ3γ8 was subjected to native-PAGE at pH 8.8 and 4°C, which separated the complex into three stained bands (Fig. 3B) . Western blot analysis (results not shown) indicated that they are, in the order of reducing mobility, pdc and the fast and the slow Tβ3γ8 bands. The identity of the fast and slow Tβ3γ8 bands as demethylated and methylated dimers, respectively, was confirmed by the ability of iPLE treatment to eliminate the slower band completely. These results indicate that pdc binding masks the methylation-associated mobility differential intrinsic to Tβ3γ8, but it does not block the access of the Tγ8 C-terminus to cleavage by iPLE. In contrast, the methyl group in the TαTβγ trimeric complex was reported to be resistant to iPLE cleavage. 30 The difference may reflect steric hindrance by hydrophobic interaction between the lipid moieties from the N-terminus of Tα and the farnesyl group of Tγ1. 
The results of Western blot analysis of pdc/Tβ3γ8 and Tβ3γ8 from native-PAGE deserve further comment. Figure 3A (left) shows that the intensities of both Tβ3 and Tγ8 immunoreactive signals for pdc/Tβ3γ8 were notably weaker than the unbound Tβ3γ8, as observed previously for the rod pdc/Tβ1γ1 complex. 24 Parallel analysis (right) by SDS-PAGE confirmed that both samples contained equal amounts of Tβ3 (and presumably Tγ8). Another control study showed that pdc/Tβ3γ8 and pdc samples containing equal amounts of pdc also gave identical immunoreactive signals for anti-pdc (results not shown), indicating that the low Tβ3 signal in the pdc/Tβ3γ8 was not caused by ineffective transblot of the trimeric complex. Although the precise reason for the difference in signal intensities is not understood, these differences do not affect our confidence in the assignment of protein bands in the native-PAGE. 
Comparison of Rod and Cone Tβγ during Native-PAGE
Rod Tβ1γ1 separated during native PAGE at pH 7.5 into two bands with an average mobility of 0.33 and 0.37, respectively (Fig. 4A) . Preincubation with iPLE removed the slow-moving band while increasing the intensity of the fast band (results not shown). The trimeric pdc/Tβ1γ1 complex migrated as a single band with an average mobility of 0.46. Thus, the relative electrophoretic mobility of pdc/Tβ1γ1 and pdc/Tβ3γ8 paralleled that of Tβ1γ1 and Tβ3γ8, respectively. Together, this indicates that pdc binding masks the methylation-associated mobility differential in either rod or cone Tβγ without affecting the intrinsic mobility differential between the rod and cone Tβγ. 
Characterization of Tβ3γ8 and pdc Interaction: Comparison to Tβ1γ1
A native-PAGE–based bandshift binding assay was developed to examine the binding between Tβ1γ1 and pdc. 24 To compare the interaction between Tβ3γ8 and pdc, equal amounts of Tβ1γ1 and Tβ3γ8 were incubated with limited but increasing amounts of pdc (Fig. 4A) . After native-PAGE at pH 7.5 and 4°C, the formation of the respective pdc/Tβγ complexes was monitored by the dose-dependent removal of the unbound Tβγ (Fig. 4B) . Tβ1γ1 and Tβ3γ8 showed similar dose–response curves, with 50% of methylated Tβγ removed by a 0.7 molar ratio of added pdc (pdc:Tβ), and 50% of the demethylated Tβγ removed by 1.5 molar ratio of added pdc. Because the abundance of the methylated and demethylated forms of Tβγ were essentially identical in both rod and cone samples, we interpret the results to indicate that the methylated Tβγ showed twofold higher affinity for pdc than the demethylated Tβγ. Thus, rod and cone Tβγ not only bind pdc with essentially identical affinities, but are affected by Tγ methylation in an identical manner. 
We have shown that the binding affinity between pdc and Tβ1γ1 is three times lower after PKA phosphorylation of pdc, but the phosphorylated pdc/Tβ1γ1 remained complexed during native-PAGE at pH 7.5 and at 4°C. 24 Because increased temperature was found to favor the dissociation, 21 we tested the effect of temperature on the migration pattern of PKA-phosphorylated pdc/Tβ1γ1 (Fig. 5) . Figure 5A shows that pdc/Tβ1γ1, at 4°C and with or without PKA phosphorylation, migrated similarly as a trimeric complex. Figure 5B shows that the migration patterns at 22°C are strikingly different. In the unphosphorylated pdc/Tβ1γ1, four bands were detected that corresponded to, in the order of increasing mobility, the Tβ1γ1-OCH3, Tβ1γ1-OH, pdc/Tβ1γ1, and pdc. The staining intensity of the pdc/Tβ1γ1 was considerably darker than those of the unbound Tβ1γ1 and pdc, indicating that most of the protein remained complexed. In the phosphorylated pdc/Tβ1γ1, four bands were also detected, but the staining intensity in the pdc/Tβ1γ1 region was greatly diminished, with concomitant increase in the staining of the Tβ1γ1 and pdc subunits, indicating dissociation of most of the phosphorylated pdc/Tβ1γ1. It was noteworthy that the unbound pdc in the phosphorylated sample was not only labeled by 32P (Fig. 5C) but also showed higher mobility than corresponding pdc in the unphosphorylated sample, consistent with an increase in the negative charges as the result of phosphorylation. Parallel analysis showed that pdc/Tβ3γ8 also underwent PKA phosphorylation-induced changes in migration pattern during native-PAGE at 22°C (Figs. 5D 5E) , suggesting that binding between phosphorylated pdc and Tβ3γ8 is also reduced by at least threefold. 
Discussion
There is intense interest in understanding the molecular mechanism of cone physiology, because cones are responsible for visual acuity and cones survive better than rods during trauma and diseases. To gain better understanding of how the cell-specific transducin contributes to the cone’s unique physiology, we determined the structure of Tγ8. In addition to confirming the entire amino acid sequence as predicted by the Gγ8 cDNA, 16 we showed that the mature Tγ8 is acetylated at the N-terminus and farnesylated and methylated at the C-terminus. This indicates that the lower membrane affinity of cone transducin is unrelated to isoprenylation of the Tβ3γ8 subunit. The modification observed at the N-terminus is also consistent with the empiric rule that peptides with an N-terminal alanine, such as in Tγ8, are usually acetylated, whereas those with an N-terminal proline, such as in Tγ1, are not. 32 The purified cone Tβ3γ8 dimer, like rod Tβ1γ1, is heterogeneous with respect to the state of methylation at the C-terminus of Tγ8. The amount of methylated Tγ8 is highest in the freshly prepared pdc/Tβ3γ8 complex and lowest in the dissociated Tβ3γ8 that has been stored for an extended period, raising the intriguing possibility that pdc may exert protection against demethylation. Loew et al. 33 showed that pdc induces minor conformational changes in Tβ1γ1, resulting in the formation of a shallow cavity in Τβ1 that partially buries the farnesyl group. The effect of pdc on the conformation at the methylation site is less clear, because the precise conformation in the crystallized Tβ1γ1 is not known. 34 Results from our iPLE experiments indicate that pdc does not block demethylation by this enzyme, but they do not rule out the possibility that pdc may slow down demethylation. 
The presence of the same pdc in rods and cones of bovine retinas has been established (Lee RH, et al., unpublished observation, 2003). In this study, we compared the binding of the rod and cone Tβγ to pdc and used the crystal structures of Tβ1γ1 and pdc/Tβ1γ1 as models to gain insight into the Tβ3γ8 and pdc interaction. It was found that both Tβγ bind pdc with the same affinity and are affected in the same manners by PKA phosphorylation of pdc and by changes in the status of methylation in the respective Tγ. The binding assay for pdc and Tβγ was developed based on the ability of the native-PAGE to separate the pdc/Tβγ complex from the unbound pdc and Tβγ. The electrophoretic mobility of Tβγ is highly sensitive to variations and other subtler changes on the surface and offers an independent parameter for evaluating the conformation of Tβγ and the topology of pdc and Tβγ interaction. Rod Tβγ shows higher mobility than cone Tβγ, consistent with their respective isoelectric points (pIs) of 3.9 and 4.1. 15 For each type of Tβγ, the demethylated form shows higher mobility than the methylated form. Pdc binding masks the methylation-associated mobility differential, but not the intrinsic rod/cone difference. Collectively, these observations indicate that (1) the pdc binding surface on cone Tβγ is the same as rod Tβγ; (2) the methylation site is situated on a conserved surface on rod and cone Tβγ, and this surface is either close to or overlaps with the pdc binding domain; and (3) away from the pdc binding domains, there exist a unique surface whose electrostatic characteristics underlies the intrinsic mobility differential between rod and cone Tβγ. These notions are entirely consistent with the three-dimensional structure of Tβ1γ1. 33 34 35 For example, the most variable segments of Tβ1 and Tγ1, located at the far N-termini of both peptides, intertwine into helixes or loops that form an exposed surface away from the pdc binding surface. In summary, our results showed that rod and cone Tβγ are identically modified at the C-terminus of the respective Tγ subunits, assume the same conformation, and interact with pdc in the same manner, indicating that pdc and Tβγ interaction does not play a major role in the specialization of rod and cone physiology. 
Hydrophobic binding through the isoprenyl group is the major mechanism by which Τβγ and, other Gβγ isoforms target cell membranes and their effector proteins. 9 10 Reversible methylation at the neighboring carboxyl group is thought to play a role in regulating isoprenylated proteins. 11 In rod outer segments, Tβ1γ1, along with the α, β-subunits of PDE and the small G protein, undergoes reversible methylation. 36 The enzymes that specifically catalyze the methylation and demethylation of the farnesylated cysteine have also been identified. 37 38 Several in vitro studies have shown that methylation increases the Tβγ-mediated activities 30 31 39 ; most discussions credited the effects to enhanced hydrophobicity and membrane binding. However, several observations from this study indicate that reversible methylation also induces conformational changes that modify the electrostatic surface of Tβγ. First, during native-PAGE the demethylated Tβγ migrated much faster than the methylated dimer, indicating large electrostatic changes that could not be explained by the difference of one negative charge on the intrinsically acidic rod or cone Τβγ. 15 Second, pdc masked the mobility differential between methylated and demethylated Tβγ, suggesting an overlap between the pdc binding domain and the surface that is electrostatically regulated by Tγ methylation. Third, the demethylated Tβγ shows twofold lower affinity for pdc than the methylated dimer, even though the crystal structure shows that the Tγ methylation site is outside of the pdc binding surface on Tβγ. 33 35 The crystal structure of Tβ1γ1 suggests a plausible mechanism for such changes. It is known that the farnesyl group and the methylation site of Tγ is surrounded by a prominent patch of positive charges formed by 11 basic residues that scatter throughout the Tβ peptide but come together in the three dimensional space. 34 35 40 This means that reversible methylation, by regulating the presence or absence of a negative charge juxtaposed at the center of domain, is poised to induce far-reaching changes in the spatial arrangement of these basic residues. Of note, this basic surface on Tβγ is not only a part of membrane binding domain but also overlaps with a highly conserved sequence motif, HIKE,42 which encompasses amino acids residues that are essential for as well as regulate Tβγ interaction with pdc and most of the Gβγ effectors identified to date (Fig. 6) . A recent report has shown that electrostatic interactions between this positively changed domain and the membranous acidic phospholipids increase the membrane partitioning of Tβγ by one order of magnitude. 40 This means that electrostatic changes induced by reversible methylation may also play a role in regulating Tβγ binding to membranes. It is noteworthy that Tβγ binding to pdc and the Tβγ-stimulated membrane-dependent GTP-S exchange by Tα 30 are similarly affected by reversible methylation, suggesting a common underlying regulatory mechanism. 
In summary, this study has presented the first evidence of methylation-induced conformational changes on a multifunctional surface domain on Tβγ that is essential for its functions. We suggest that these changes are essential mechanisms by which reversible methylation regulates Tβγ activities. 
 
Figure 1.
 
RP-HPLC of rod and cone pdc/Tβγ complex and the molecular weight reconstructs of Tγ. The pdc/Tβ1γ1 (A) or the pdc/Tβ3γ8 (B) was subjected to RP-HPLC, and the elution profile was monitored by absorbance at 280 nm. Western blot analyses of individual fractions showed that absorbance peak Gr contained Tγ1, peaks Gc1 and Gc2 contained Tγ8, and peak pdc contained pdc (results not shown). Peaks Gr, Gc1, and Gc2 were subjected to ESIMS. The resultant mass spectra were deconvoluted to reveal the masses and the relative abundance of molecules in each peak (A, B, insets).
Figure 1.
 
RP-HPLC of rod and cone pdc/Tβγ complex and the molecular weight reconstructs of Tγ. The pdc/Tβ1γ1 (A) or the pdc/Tβ3γ8 (B) was subjected to RP-HPLC, and the elution profile was monitored by absorbance at 280 nm. Western blot analyses of individual fractions showed that absorbance peak Gr contained Tγ1, peaks Gc1 and Gc2 contained Tγ8, and peak pdc contained pdc (results not shown). Peaks Gr, Gc1, and Gc2 were subjected to ESIMS. The resultant mass spectra were deconvoluted to reveal the masses and the relative abundance of molecules in each peak (A, B, insets).
Figure 2.
 
Mass peptide mapping of Tγ8. Tγ8 was digested with either Arg-C or Lys-C and the masses of the resultant fragments (identified by R’s and L’s, respectively) were determined by ESIMS. Each fragment was identified by its mass and mapped to the proposed sequence for the mature Tγ8. The location and the observed and the theoretical masses (in parenthesis) of each fragment are indicated by the brackets. The R2′ fragment is the demethylated form of R2. The posttranslationally added moieties are indicated by italic notations, including the acetyl (Ac), the farnesyl (C 12 H 25 ), and the methyl (CH 3 ) groups. The sequence in parentheses identifies the amino acid residues that are removed from the predicted nascent peptide by posttranslational modification.
Figure 2.
 
Mass peptide mapping of Tγ8. Tγ8 was digested with either Arg-C or Lys-C and the masses of the resultant fragments (identified by R’s and L’s, respectively) were determined by ESIMS. Each fragment was identified by its mass and mapped to the proposed sequence for the mature Tγ8. The location and the observed and the theoretical masses (in parenthesis) of each fragment are indicated by the brackets. The R2′ fragment is the demethylated form of R2. The posttranslationally added moieties are indicated by italic notations, including the acetyl (Ac), the farnesyl (C 12 H 25 ), and the methyl (CH 3 ) groups. The sequence in parentheses identifies the amino acid residues that are removed from the predicted nascent peptide by posttranslational modification.
Figure 3.
 
Native-PAGE of Tβ3γ8: the effect of pdc binding and methylation. (A) Effect of pdc binding: Pdc/Tβ3γ8 (sample 1) and Tβ3γ8 (sample 2), containing equal amounts of Tβ3 were subjected to native-PAGE at pH 7.5 and 4°C or to SDS-PAGE followed by CBB staining. The identity of individual bands was confirmed by Western blot analysis with antisera against Tβ3, Tγ8, or pdc. (mb), mobility of protein bands during Native-PAGE. (B) Effect of methylation: Pdc/Tβ3γ8 (sample 1) and Tβ3γ8 (sample 2) were incubated with or without iPLE and were subjected to native-PAGE at pH 7.5 or 8.8, followed by CBB staining.
Figure 3.
 
Native-PAGE of Tβ3γ8: the effect of pdc binding and methylation. (A) Effect of pdc binding: Pdc/Tβ3γ8 (sample 1) and Tβ3γ8 (sample 2), containing equal amounts of Tβ3 were subjected to native-PAGE at pH 7.5 and 4°C or to SDS-PAGE followed by CBB staining. The identity of individual bands was confirmed by Western blot analysis with antisera against Tβ3, Tγ8, or pdc. (mb), mobility of protein bands during Native-PAGE. (B) Effect of methylation: Pdc/Tβ3γ8 (sample 1) and Tβ3γ8 (sample 2) were incubated with or without iPLE and were subjected to native-PAGE at pH 7.5 or 8.8, followed by CBB staining.
Figure 4.
 
Comparison of the pdc binding affinity of Tβ1γ1 and Tβ3γ8. (A) Two micrograms each of Tβ1γ1 (lanes 1 to 5) and Τβ3γ8 (lanes 6 to 10) were incubated with indicated amounts of pdc before being subjected to native-PAGE at pH 7.5 and at 4°C. The formation of pdc/Tβγ complex and the concomitant removal of unbound Tβγ dimers were visualized by CBB staining. (B) The binding of pdc to Tβ1γ1 and Tβ3γ8 was monitored by dose-dependent removal of the Tβγ dimers. The amounts of individual unbound dimers were quantified by densitometric scanning and expressed as a percentage of corresponding signals detected in the absence of added pdc (control). (○) Tβ1γ1-OCH3; (□) Tβ1γ1-OH; (•) Tβ3γ8-OCH3; (▪) Tβ3γ8-OH.
Figure 4.
 
Comparison of the pdc binding affinity of Tβ1γ1 and Tβ3γ8. (A) Two micrograms each of Tβ1γ1 (lanes 1 to 5) and Τβ3γ8 (lanes 6 to 10) were incubated with indicated amounts of pdc before being subjected to native-PAGE at pH 7.5 and at 4°C. The formation of pdc/Tβγ complex and the concomitant removal of unbound Tβγ dimers were visualized by CBB staining. (B) The binding of pdc to Tβ1γ1 and Tβ3γ8 was monitored by dose-dependent removal of the Tβγ dimers. The amounts of individual unbound dimers were quantified by densitometric scanning and expressed as a percentage of corresponding signals detected in the absence of added pdc (control). (○) Tβ1γ1-OCH3; (□) Tβ1γ1-OH; (•) Tβ3γ8-OCH3; (▪) Tβ3γ8-OH.
Figure 5.
 
Effect of PKA phosphorylation on the binding of pdc to Tβ3γ8. Four micrograms each of pdc/Tβ1γ1 and pdc/Tβ3γ8 were incubated with PKA with or without [32P] adenosine triphosphate (ATP). At the end of incubation samples were subjected to native-PAGE at pH 7.5 and at either 4°C (A) or 22°C (BE). After CBB staining, the gels were dried and autoradiographed to locate the 32P-labeled proteins. The designation of various Tβγ and pdc/Tβγ bands is as in the previous figures. Pdc and 32P-pdc are unphosphorylated and 32P-labeled pdc, respectively.
Figure 5.
 
Effect of PKA phosphorylation on the binding of pdc to Tβ3γ8. Four micrograms each of pdc/Tβ1γ1 and pdc/Tβ3γ8 were incubated with PKA with or without [32P] adenosine triphosphate (ATP). At the end of incubation samples were subjected to native-PAGE at pH 7.5 and at either 4°C (A) or 22°C (BE). After CBB staining, the gels were dried and autoradiographed to locate the 32P-labeled proteins. The designation of various Tβγ and pdc/Tβγ bands is as in the previous figures. Pdc and 32P-pdc are unphosphorylated and 32P-labeled pdc, respectively.
Figure 6.
 
Alignment of the HIKE motif in Tβ1 with amino acid residues that contact pdc and form the positive-charged domain. The amino acid sequence between residues 27 and 101 of bovine Tβ1 is depicted by the one-letter code. The core of the HIKE sequence motif resides between residues 27 and 77. 41 (+), amino acids that are completely conserved among all HIKE-containing molecules and across species; (⋄) residues that directly contact pdc 33 35 ; ( Image not available ) residues that are part of the 11 basic residues forming the positive-charged domain that surrounds the C-terminus of Tγ and regulates Tβγ binding to membranes. 40 Blades 1 and 7 identify residues in this segment of Tβ1 that fold into blade 1 and a part of blade 7, respectively, in the β propeller structure of the Tβγ dimer (nomenclature according to Gaudet et al. 35 ).
Figure 6.
 
Alignment of the HIKE motif in Tβ1 with amino acid residues that contact pdc and form the positive-charged domain. The amino acid sequence between residues 27 and 101 of bovine Tβ1 is depicted by the one-letter code. The core of the HIKE sequence motif resides between residues 27 and 77. 41 (+), amino acids that are completely conserved among all HIKE-containing molecules and across species; (⋄) residues that directly contact pdc 33 35 ; ( Image not available ) residues that are part of the 11 basic residues forming the positive-charged domain that surrounds the C-terminus of Tγ and regulates Tβγ binding to membranes. 40 Blades 1 and 7 identify residues in this segment of Tβ1 that fold into blade 1 and a part of blade 7, respectively, in the β propeller structure of the Tβγ dimer (nomenclature according to Gaudet et al. 35 ).
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Figure 1.
 
RP-HPLC of rod and cone pdc/Tβγ complex and the molecular weight reconstructs of Tγ. The pdc/Tβ1γ1 (A) or the pdc/Tβ3γ8 (B) was subjected to RP-HPLC, and the elution profile was monitored by absorbance at 280 nm. Western blot analyses of individual fractions showed that absorbance peak Gr contained Tγ1, peaks Gc1 and Gc2 contained Tγ8, and peak pdc contained pdc (results not shown). Peaks Gr, Gc1, and Gc2 were subjected to ESIMS. The resultant mass spectra were deconvoluted to reveal the masses and the relative abundance of molecules in each peak (A, B, insets).
Figure 1.
 
RP-HPLC of rod and cone pdc/Tβγ complex and the molecular weight reconstructs of Tγ. The pdc/Tβ1γ1 (A) or the pdc/Tβ3γ8 (B) was subjected to RP-HPLC, and the elution profile was monitored by absorbance at 280 nm. Western blot analyses of individual fractions showed that absorbance peak Gr contained Tγ1, peaks Gc1 and Gc2 contained Tγ8, and peak pdc contained pdc (results not shown). Peaks Gr, Gc1, and Gc2 were subjected to ESIMS. The resultant mass spectra were deconvoluted to reveal the masses and the relative abundance of molecules in each peak (A, B, insets).
Figure 2.
 
Mass peptide mapping of Tγ8. Tγ8 was digested with either Arg-C or Lys-C and the masses of the resultant fragments (identified by R’s and L’s, respectively) were determined by ESIMS. Each fragment was identified by its mass and mapped to the proposed sequence for the mature Tγ8. The location and the observed and the theoretical masses (in parenthesis) of each fragment are indicated by the brackets. The R2′ fragment is the demethylated form of R2. The posttranslationally added moieties are indicated by italic notations, including the acetyl (Ac), the farnesyl (C 12 H 25 ), and the methyl (CH 3 ) groups. The sequence in parentheses identifies the amino acid residues that are removed from the predicted nascent peptide by posttranslational modification.
Figure 2.
 
Mass peptide mapping of Tγ8. Tγ8 was digested with either Arg-C or Lys-C and the masses of the resultant fragments (identified by R’s and L’s, respectively) were determined by ESIMS. Each fragment was identified by its mass and mapped to the proposed sequence for the mature Tγ8. The location and the observed and the theoretical masses (in parenthesis) of each fragment are indicated by the brackets. The R2′ fragment is the demethylated form of R2. The posttranslationally added moieties are indicated by italic notations, including the acetyl (Ac), the farnesyl (C 12 H 25 ), and the methyl (CH 3 ) groups. The sequence in parentheses identifies the amino acid residues that are removed from the predicted nascent peptide by posttranslational modification.
Figure 3.
 
Native-PAGE of Tβ3γ8: the effect of pdc binding and methylation. (A) Effect of pdc binding: Pdc/Tβ3γ8 (sample 1) and Tβ3γ8 (sample 2), containing equal amounts of Tβ3 were subjected to native-PAGE at pH 7.5 and 4°C or to SDS-PAGE followed by CBB staining. The identity of individual bands was confirmed by Western blot analysis with antisera against Tβ3, Tγ8, or pdc. (mb), mobility of protein bands during Native-PAGE. (B) Effect of methylation: Pdc/Tβ3γ8 (sample 1) and Tβ3γ8 (sample 2) were incubated with or without iPLE and were subjected to native-PAGE at pH 7.5 or 8.8, followed by CBB staining.
Figure 3.
 
Native-PAGE of Tβ3γ8: the effect of pdc binding and methylation. (A) Effect of pdc binding: Pdc/Tβ3γ8 (sample 1) and Tβ3γ8 (sample 2), containing equal amounts of Tβ3 were subjected to native-PAGE at pH 7.5 and 4°C or to SDS-PAGE followed by CBB staining. The identity of individual bands was confirmed by Western blot analysis with antisera against Tβ3, Tγ8, or pdc. (mb), mobility of protein bands during Native-PAGE. (B) Effect of methylation: Pdc/Tβ3γ8 (sample 1) and Tβ3γ8 (sample 2) were incubated with or without iPLE and were subjected to native-PAGE at pH 7.5 or 8.8, followed by CBB staining.
Figure 4.
 
Comparison of the pdc binding affinity of Tβ1γ1 and Tβ3γ8. (A) Two micrograms each of Tβ1γ1 (lanes 1 to 5) and Τβ3γ8 (lanes 6 to 10) were incubated with indicated amounts of pdc before being subjected to native-PAGE at pH 7.5 and at 4°C. The formation of pdc/Tβγ complex and the concomitant removal of unbound Tβγ dimers were visualized by CBB staining. (B) The binding of pdc to Tβ1γ1 and Tβ3γ8 was monitored by dose-dependent removal of the Tβγ dimers. The amounts of individual unbound dimers were quantified by densitometric scanning and expressed as a percentage of corresponding signals detected in the absence of added pdc (control). (○) Tβ1γ1-OCH3; (□) Tβ1γ1-OH; (•) Tβ3γ8-OCH3; (▪) Tβ3γ8-OH.
Figure 4.
 
Comparison of the pdc binding affinity of Tβ1γ1 and Tβ3γ8. (A) Two micrograms each of Tβ1γ1 (lanes 1 to 5) and Τβ3γ8 (lanes 6 to 10) were incubated with indicated amounts of pdc before being subjected to native-PAGE at pH 7.5 and at 4°C. The formation of pdc/Tβγ complex and the concomitant removal of unbound Tβγ dimers were visualized by CBB staining. (B) The binding of pdc to Tβ1γ1 and Tβ3γ8 was monitored by dose-dependent removal of the Tβγ dimers. The amounts of individual unbound dimers were quantified by densitometric scanning and expressed as a percentage of corresponding signals detected in the absence of added pdc (control). (○) Tβ1γ1-OCH3; (□) Tβ1γ1-OH; (•) Tβ3γ8-OCH3; (▪) Tβ3γ8-OH.
Figure 5.
 
Effect of PKA phosphorylation on the binding of pdc to Tβ3γ8. Four micrograms each of pdc/Tβ1γ1 and pdc/Tβ3γ8 were incubated with PKA with or without [32P] adenosine triphosphate (ATP). At the end of incubation samples were subjected to native-PAGE at pH 7.5 and at either 4°C (A) or 22°C (BE). After CBB staining, the gels were dried and autoradiographed to locate the 32P-labeled proteins. The designation of various Tβγ and pdc/Tβγ bands is as in the previous figures. Pdc and 32P-pdc are unphosphorylated and 32P-labeled pdc, respectively.
Figure 5.
 
Effect of PKA phosphorylation on the binding of pdc to Tβ3γ8. Four micrograms each of pdc/Tβ1γ1 and pdc/Tβ3γ8 were incubated with PKA with or without [32P] adenosine triphosphate (ATP). At the end of incubation samples were subjected to native-PAGE at pH 7.5 and at either 4°C (A) or 22°C (BE). After CBB staining, the gels were dried and autoradiographed to locate the 32P-labeled proteins. The designation of various Tβγ and pdc/Tβγ bands is as in the previous figures. Pdc and 32P-pdc are unphosphorylated and 32P-labeled pdc, respectively.
Figure 6.
 
Alignment of the HIKE motif in Tβ1 with amino acid residues that contact pdc and form the positive-charged domain. The amino acid sequence between residues 27 and 101 of bovine Tβ1 is depicted by the one-letter code. The core of the HIKE sequence motif resides between residues 27 and 77. 41 (+), amino acids that are completely conserved among all HIKE-containing molecules and across species; (⋄) residues that directly contact pdc 33 35 ; ( Image not available ) residues that are part of the 11 basic residues forming the positive-charged domain that surrounds the C-terminus of Tγ and regulates Tβγ binding to membranes. 40 Blades 1 and 7 identify residues in this segment of Tβ1 that fold into blade 1 and a part of blade 7, respectively, in the β propeller structure of the Tβγ dimer (nomenclature according to Gaudet et al. 35 ).
Figure 6.
 
Alignment of the HIKE motif in Tβ1 with amino acid residues that contact pdc and form the positive-charged domain. The amino acid sequence between residues 27 and 101 of bovine Tβ1 is depicted by the one-letter code. The core of the HIKE sequence motif resides between residues 27 and 77. 41 (+), amino acids that are completely conserved among all HIKE-containing molecules and across species; (⋄) residues that directly contact pdc 33 35 ; ( Image not available ) residues that are part of the 11 basic residues forming the positive-charged domain that surrounds the C-terminus of Tγ and regulates Tβγ binding to membranes. 40 Blades 1 and 7 identify residues in this segment of Tβ1 that fold into blade 1 and a part of blade 7, respectively, in the β propeller structure of the Tβγ dimer (nomenclature according to Gaudet et al. 35 ).
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