Mol Biol Rep DOI 10.1007/s11033-011-1225-0
Glucagon like-peptide-1 receptor is covalently modified by endogenous mono-ADP-ribosyltransferase Matjazˇ Dezˇelak • Aljosˇa Bavec
Received: 21 December 2010 / Accepted: 20 June 2011 Ó Springer Science+Business Media B.V. 2011
Abstract Our previous study revealed a mono-ADPribosyltransferase mediated in vitro mono-ADP-ribosylation of IC3 peptide, a peptide with sequence corresponded to third intracellular loop of glucagon like-peptide-1 (GLP1) receptor. Furthermore, Arg348 was shown to be modified amino acid residue although its mutation did not eliminate mono-ADP-ribosylation completely. In order to further study the signaling mechanisms of GLP-1 receptor, we took on lease a possibility that an alternative site of enzymatic modification exist so mono-ADP-ribosylation of Cys341 was hypothesized. The results confirmed both Arg348 and Cys341 as a site of mono-ADP-ribosylation where Arg348 is modified predominantly. Sum of monoADP-ribosylation rate of both single IC3 mutants coincided with IC3 rate. What is in vivo role of Cys341 mono-ADPribosylation is entirely speculative but our study represents an important step toward a complete understanding of signaling via GLP-1 receptor. Keywords Glucagon like peptide-1 receptor Synthetic receptor-loop peptides ADP-ribosylation Posttranslational modification Diabetes Abbreviations GLP-1 Glucagon-like peptide-1 CHO Chinese hamster ovary IC3 Third intracellular loop-derived peptide IC3(R348A) Arg to Ala mutant of IC3
M. Dezˇelak A. Bavec (&) Institute of Biochemistry, Faculty of Medicine, University of Ljubljana, Vrazov trg 2, 1000 Ljubljana, Slovenia e-mail:
[email protected]
IC3(C341A) mono-ART
Cys to Ala mutant of IC3 Mono-ADP-ribosyltransferase
Introduction The glucagon-like peptide-1 receptor (GLP-1 receptor) is a member of secretin/vasointestinal peptide receptor family B [1–3]. GLP-1 receptor mediates the effects of GLP-1 on peripheral tissues, such as the stimulation of glucose-induced insulin secretion in b-cells [4, 5], arterial blood pressure and heart rate [6], and in the central nervous system it exerts effects on food and water intake [7], and enhances associative and spatial learning [8]. The ability of GLP-1 to induce insulin secretion in dependence on high glucose levels renders this component potentially useful in the treatment of non-insulindependent diabetes mellitus type 2 [9]. Different types of a subunits of G-proteins are coupled to GLP-1 receptor in Chinese hamster ovary cells (CHO) [10] and sf9 cells [11]. Importance of the third intracellular (IC3) loop in activation of G-proteins and adenylyl cyclase was revealed by point and block deletion mutations of the receptor and by peptide based interaction mapping of the receptor [11–14]. Diverse role of three intracellular loops of GLP-1 receptor in G-protein coupling was also shown; third loop is the main switch that mediates signaling via GLP-1 receptor to G-proteins, while first and second loops discriminate between different types of G-proteins [11]. Mono-ADP-ribosylation, like protein phosphorylation [15], has all the characteristics of a mechanism important for cell regulation. In case of b subunit of G proteins this reaction is catalyzed by an arginine-specific, plasma membrane associated, mono-ADP-ribosyltransferase (monoART) [16]. Enzyme specifically modifies arginine at position
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129, a residue in the effector-binding domain of the b subunit [16]. Mono-ADP-ribosylation of this residue prevents b subunit-dependent modulation of effectors such as type 1 adenylyl cyclase [17], phosphoinositide 3-kinase-c and phospholipase C-b2 [18]. The modified b subunit is further a substrate for a cytosolic mono-ADP-ribosylhydrolase that releases the bound ADP-ribose from the mono-ADP-ribosylated protein [17]. These activities are part of the intracelular regulation of the bc dimer function. In this study, our primary aim was to proceed with the investigation of functional coupling between GLP-1 receptor and endogenous mono-ART in CHO cells. Our previous study [19] revealed an in vitro mono-ADP-ribosylation of IC3 peptide, a peptide with sequence corresponded to third intracellular loop of GLP-1 receptor. Proposed site for covalent modification was Arg348 therefore we further tested a site-specific mutant of IC3, named IC3(R348A). Its effect on mono-ADP-ribosylation of b subunit and its own mono-ADP-ribosylation rate confirmed Arg348 as a modified amino acid residue but suggested existence of an alternative site of enzymatic modification as well. Besides arginine, cysteine is another well known amino acid residue being mono-ADP-ribosylated, as was shown for cell surface mono-ADP-ribosylated proteins on human monocytes [20], glutamate dehydrogenase isozymes [21] and GTP-binding protein Gi alpha subunits [22]. Thus we hypothesized Cys341 as a most probable candidate. In this manner we synthesized a 23 amino acid peptide IC3 corresponding to sequence derived from the third intracellular loop of GLP-1 receptor (329–351), and three mutant peptides, i.e. IC3(R348A), IC3(C341A) and IC3(R348A, C341A). We followed the endogenous monoART-mediated mono-ADP-ribosylation of G-proteins b subunit in absence or presence of different concentrations of IC3, IC3(C341A) and IC3(R348A, C341A) peptides. Additionally, we followed mono-ADP-ribosylation of peptides at different concentrations by mono-ART. Expected results arose where most important information is that sum rate of IC3(R348A) and IC3(C341A) mono-ADPribosylation coincided with IC3 mono-ADP-ribosylation rate. IC3(R348A, C341A) peptide undergo mono-ADPribosylation at very low rate.
Methods and materials Cell cultures CHO cells were grown as a monolayer culture in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 0.39% NaHCO3, 58 mg/l proline, 53 mg/l aspartic acid, 59 mg/l glutamic acid,
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60 mg/l asparagine, 100 units/ml penicillin, 100 lg/ml streptomycin at 37°C in a 5% CO2 atmosphere. Peptide synthesis Peptide IC3 with sequence CIVIAKLKANLMCKTDIKCR LAK, and three mutant peptides, i.e. IC3(R348A) with sequence CIVIAKLKANLMCKTDIKCALAK, IC3(C341A) with sequence CIVIAKLKANLMAKTDIKCRLAK) and IC3(R348A, C341A) with sequence (CIVIAKLKANLMA KTDIKCALAK) were synthesized according to the protocol described previously [14]. Plasma membrane preparation CHO plasma membranes were prepared as described previously [23] with minor modification. Briefly, cells (approximately 2 9 108) were washed in Hanks’ balanced salt solution (HBSS) without Ca2? and Mg2?, detached with hypotonic buffer, and homogenized in Teflon/glass Potter homogenizer. After centrifugation of homogenate at 3009 g, crude membranes were collected from the supernatant by a 20 min centrifugation at 420009 g. The membranes were further purified by aqueous two-phase system with dextran and polyethylene glycol. All procedures were carried out at 4°C. The concentration of proteins in plasma membranes was determined with Biorad Protein Assay at 595 nm. Mono-ADP-ribosylation assay ADP-ribosylation followed the incorporation of [32P] ADPribose into the b subunit of heterotrimeric G-protein and was performed with 5 lg of CHO plasma membrane preparation as described previously [23] with minor modification. The 50 ll of plasma membranes in the ADPribosylation buffer (50 mM potassium phosphate buffer (pH = 7.5), 0.5 mM MgCl2, 4 mM dithiothreitol, 5 9 106 cpm or 2.3 lCi [32P]NAD? (specific activity = 1000 Ci/ mmol) and 6 lM b-NAD?) were incubated at 37°C for 1 h with the indicated peptides. The ADP-ribosylation reactions were stopped by diluting the samples with 50 ll Laemmli buffer, then boiled, and analyzed by 11% SDS-PAGE followed by electroblot onto nitrocellulose membrane. The radioactivity incorporated into the membrane proteins was detected and quantified by Instant Imager Packard (manual integration and subtraction of background) or probed with specific anti-b antibodies (1:1000, Santa Cruz). Immunoblotting analysis Immunoblotting analysis was prepared as described previously [24], but again with minor modifications. After the run
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on SDS-PAGE (16 cm long), proteins were transferred from the gel onto nitrocellulose membrane with a constant current of 400 mA for 5 h. The blot was first incubated for 1 h in blocking buffer (2% bovine serum albumin in TBS; 20 mM Tris–HCl, 500 mM NaCl; pH = 7.5). The blocking buffer was then replaced by a buffer TTBS (0.05% Tween 20 in TBS) and incubated with the primary polyclonal rabbit IgG anti-b antibodies (T20, 1:1000, Santa Cruz) at room temperature for 2 h. The primary antibodies were washed three-times in TTBS buffer for 10 min and incubated with secondary peroxidase-conjugated goat anti-rabbit IgG antibodies (1:1000, Calbiochem) at room temperature for another 1 h. After the wash in TTBS buffer for three times for 10 min and once in TBS for 10 min, the immunoreactive proteins caught on the blot were exposed for 15–60 s to Kodak X-Omat film using ECL chemiluminescence detection kit (Amersham Pharmacia Biotech, RPN 2106). Statistical methods Data are expressed as means ± SD of at least three independent experiments and the statistical analysis were carried out by PRISM5 computer program (GraphPad Software, USA), which was also used for graphical presentation of the results.
characterized substrate by date of this arginine-specific mono-ART is b subunit of heterotrimeric G-proteins in plasma membranes [17]. At first, activity of aforementioned endogenous mono-ART was followed by monoADP-ribosylation of b-subunit in the absence or presence of different concentrations of IC3(C341A) peptide. It was previous shown [19] that IC3 act as a competitive inhibitor where Arg348 is obviously main amino acid residue that undergo mono-ADP-ribosylation. However, mono-ADPribosylation of IC3(R348A) is still significant therefore an alternative mono-ADP-ribosylation site was proposed, in particular Cys341. The mono-ADP-ribosylation protein bands with an apparent molecular mass &36 kDa corresponded to b subunit of G-proteins and its mono-ADP-ribosylation was carefully quantified (Fig. 1a, b). Bands additionally confirmed by ECL Western blot analysis using anti-b antibodies were also used as calibrator proteins (Fig. 1c). IC3(C341A) inhibits mono-ADP-ribosylation of b subunit in dose-dependent manner (Fig. 1a, b), which is in accordance with our previous work [19] where we showed that IC3 peptide with intact Arg348 act as a competitive inhibitor of mono-ADP-ribosylation of b subunit.
Materials Chinese hamster ovary cells (CHO-K1) were obtained from ECACC, UK. [32P]NAD?, ESL kit and Dextran 500 were supplied from Amersham Pharmacia Biotech, UK. HBSS was provided by Gibco, UK and fetal bovine serum was from Biochrom (Germany). DMEM, acrylamide/bis-acrylamide and other electrophoresis reagents were from Eurobio, France. Nitrocellulose transfer membranes were from Schleicher and Schuell, Germany. Ultima Gold scintillation liquid was from Packard, USA. Polyethylene glycol 4000 was from Merck (USA). Rabbit polyclonal anti-b (T-20) was from Santa Cruz Biotechnology (USA). Goat anti-rabbit IgG horseradish peroxidase conjugates were from Calbiochem, USA. All other chemicals were from Sigma Aldrich Co, USA and were of analytical grade.
Results Effect of IC3(C341A) peptide on mono-ADPribosylation of b subunit of G-proteins by mono-ART from CHO cells Endogenous mono-ART is well expressed in CHO cells and localized at cytosolic side of plasma membrane as a GPI anchored peripheral protein [17]. The best
Fig. 1 Effect of IC3 and IC3(C341A) peptides on mono-ADPribosylation of b subunit of G-proteins by mono-ART from CHO cells. a Quantitative analysis of mono-ADP-ribosylation of b subunit by IC3 (white bars) or IC3(C341A) (grey bars). Each bar of IC3 series with the indicated standard deviation represents the mean value of four independent experiments (ANOVA: F(11, 36) = 18.86; P \ 0,0001). Tukey–Kramer multiple comparison test did not reveal any significant differences between groups of interest, P [ 0.05 was not considered significant. C—control (no peptide). b Analysis of endogenous mono-ADP-ribosylation of b subunit with SDS-PAGE and proteins transfer from the gel onto nitrocellulose membrane followed by the detection of radioactivity with IC3 (white) or IC3(C341A) (grey). c Western blot analysis of b subunit with anti-b antibodies
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Mono-ADP-ribosylation begins to decrease significantly between 25 and 50 lM and it decreases further at 100 lM (Fig. 1a, b). Furthermore, we did not observe any significant differences between inhibitory effects of IC3 and IC3(C341A) on mono-ADP-ribosylation of b subunit. Effect of IC3(R348A,C341A) peptide on mono-ADPribosylation of b subunit of G-proteins by mono-ART from CHO cells If Arg348 is the main amino acid residue exerting inhibitory effect on mono-ADP-ribosylation of b subunit during the process of competitive inhibition and Cys341 has no effect on it, IC3(R348A, C341A) peptide should show similar effect on mono-ADP-ribosylation of b subunit as does IC3(R348A) peptide. Again, mono-ADP-ribosylation protein bands with an apparent molecular mass &36 kDa corresponded to b subunit of G-proteins and its mono-ADP-ribosylation was carefully quantified (Fig. 2a, b). Bands were additionally confirmed by ECL Western blot analysis using anti-b antibodies (Fig. 2c). Indeed, double-mutant of IC3, IC3(R348A, C341A), showed no significant effect on mono-ADP-ribosylation of b subunit, similar as IC3(R348A) peptide [19]. These
Fig. 2 Effect of IC3(R348A,C341A) peptide on mono-ADP-ribosylation of b subunit of G-proteins by mono-ART from CHO cells. a Quantitative analysis of mono-ADP-ribosylation of b subunit. Each bar with the indicated standard deviation represents the mean value of three independent experiments. ANOVA: F(5, 12) = 3.512; P = 0.25. Tukey–Kramer multiple comparison test did not reveal any significant differences between groups of interest, P [ 0.05 was not considered significant. C—control (no peptide). b Analysis of endogenous mono-ADP-ribosylation of b subunit with SDS-PAGE and proteins transfer from the gel onto nitrocellulose membrane followed by the detection of radioactivity. c Western blot analysis of b subunit with anti-b antibodies
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results are consistent with Arg348 residue being the primary mono-ADP-ribosylated site during the competitive inhibition process and Cys341 having no measurable effect. Mono-ADP-ribosylation of IC3, IC3(R348A), IC3(C341A) and IC3(R348A, C341A) peptides by endogenous mono-ART from plasma membrane of CHO cells We performed mono-ADP-ribosylation assay with the increasing concentrations of IC3, IC3(R348A), IC3(C341A) and IC3(R348A, C341A) peptides. Mono-ADP-ribosylation protein bands with an apparent molecular mass &3.0 kDa corresponded to particular peptide and its mono-ADP-ribosylation was carefully quantified (Fig. 3a, b). Amount of b subunit during the experiment was unchanged (Fig. 3c). Mono-ADP-ribosylation rate of peptides in general increases with increasing peptide concentration although magnitudes vary considerably (Fig. 3a, b). As hypothesized,
Fig. 3 Mono-ADP-ribosylation of IC3, IC3(R348A), IC3(C341A) and IC3(R348A, C341A) peptides by endogenous mono-ART from plasma membrane of CHO cells. a Quantitative analysis of monoADP-ribosylation of IC3 (white bars), IC3(C341A) (grey bars), IC3(R348A) (white patterned bars) and IC3(R348A, C341A) (grey patterned bars). Each bar with the indicated standard deviation represents the mean value of three independent experiments. b Analysis of endogenous mono-ADP-ribosylation of IC3 (white), IC3(C341A) (grey), IC3(R348A) (white patterned), and IC3(R348A, C341A) (grey patterned) with SDS-PAGE and proteins transfer from the gel onto nitrocellulose membranes followed by the detection of radioactivity. c Western blot analysis of b subunit with anti-b antibodies. Due to the comparable expression rate among experiments with different peptides only one blot is showed
Mol Biol Rep Table 1 Mono-ADP-ribosylation rate of IC3 peptide and sum of IC3(R348A) and IC3(C341A) peptides together with standard deviations at different concentrations Concentration (lM)
Peptide IC3 (% of 1 lM)
IC3(R348A) ? IC3(C341A) (% of 1 lM)
25
803 ± 50
823 ± 52
50
1321 ± 106
1176 ± 67
100
2025 ± 185
1677 ± 106
mono-ADP-ribosylation of IC3(C341) peptide occurs, indeed. On the other hand, modification of IC3(R348A, C341A) is not completely lost, as supposed. Some other type of [32P] b-NAD? or [32P] ADP-ribose binding exists, although it is very low (Fig. 3a, b). The decline of mono-ADP-ribosylation rate of singlemutant peptides is between two and three times as compared to IC3, but it is always smaller in case of IC3(C341) peptide which suggests Arg348 as the predominant site of modification. That mono-ADP-ribosylation rate of IC3 peptide represents a sum of IC3(R348A) and IC3(C341) peptides rates (Table 1) is another important accordance with proposed two-site modification of IC3 peptide.
Discussion The more complex a role of particular bioactive molecule is the more important is its fine activity regulation. In case of GLP-1 receptor there is yet well known regulation via homologous and heterologous desensitization by receptor phosphorylation [15, 25] and lately proposed mono-ADPribosylation of the third intracellular loop [19]. Primary aim of this study was to determine wheather Cys341 in a peptide whose sequence correspond to the third intracellular loop of GLP-1 receptor is covalently modified by mono-ART and, if this is the case, does it affect the monoADP-ribosylation of the b subunit. Several studies have shown that synthetic peptides with the sequence derived from the predicted intracellular loops of G-protein coupled receptors, e.g. a2-adrenergic [26], D2 [27], follicle-stimulating hormone [28], and GLP-1 receptors [11, 29], can mimic the effect of activation of these receptors acting at the specific receptor-G-protein interface site. Such synthetic peptides are therefore useful tool for studying receptor-G-protein interaction [30]. Third intracellular loop IC3 of GLP-1 receptor works as competitive substrate for mono-ART in an enzymatic process which reduces mono-ADP-ribosylation of b subunit. Arg348 in IC3 is ADP-ribosylated by mono-ART [19]. MonoADP-ribosylation of b subunit is dependent on IC3(C341A) peptide concentration (Fig. 1a, b). An inhibition occurs at
concentration of 25 lM and higher and that result confirm Arg348 residue being the site responsible for inhibition of mono-ADP-ribosylation of b subunit [19]. In addition, this result exclude the involvement of Cys341 in this process since inhibitory effects by IC3 and IC3(C341A) peptides are comparable, as are comparable inhibitory effects of IC3(R348A) and IC3(R348A,C341A) peptides (Fig. 2a, b). However, there is still possibility that inhibition due to the mono-ADP-ribosylation of Cys341 residue occurs but it is neutralized by another, opposite acting process. As has been shown [14], IC3(R348A) peptide has—that depends upon a subunit type—either the same rate of activation or it activates G proteins more efficiently than the IC3. In case of efficient G protein activation there is consequential higher amount of free bc dimer released by IC3 (R348A). Since b subunit in bc dimer is the best substrate for mono-ADP ribosylation by endogenous mono-ART, the increasing IC3 concentrations should raise the amount of mono-ADP-ribosylated b subunit via raising the amount of the substrate. That is why an effect of competitive inhibition by IC3(R348A) peptide might be neutralized due to an excessive mono-ADP-ribosylation of b subunit. Additionally, mono-ADP-ribosylation of Cys341 residue is significantly lower than that of Arg348 (Fig. 3) so its inhibitory effect on mono-ADP-ribosylation of b subunit could be easily hidden. There is another confirmation that the presence of Arg348 residue is the main amino acid residue involved in mono-ADP-ribosylation. Experiments with presence of different concentrations of IC3(R348A, C341A) peptide showed no statistical significant inhibition of mono-ADPribosylation of b subunit (Fig. 2a, b). Similar results were also obtained in experiment with IC3(R348A) peptide [14]. Although in case of IC3(R348A) peptide there is still possibility of competitive inhibition, this is not the case in experiment with double mutant since IC3(R348A, C341A) peptide did not show any increase of G protein activation [14]. Finally, actual peptide mono-ADP-ribosylation was investigated. Both IC3(R348A) and IC3(C341A) exerted significant level of mono-ADP-ribosylation (Fig. 3a, b) although they never reached 50% of IC3 peptide level. Sum of their mono-ADP-ribosylation was comparable with one of IC3 peptide (Table 1). Radioactive signal of IC3(R348A, C341A) was not totally diminished, although its intensity was relatively insignificant compared to other three peptides (Fig. 3a, b) and could represents an experimental noise. Mono-ADP-ribosylation of Cys341 indeed occurs but it has no measurable inhibitory effect on mono-ADPribosylation of b subunit. Besides arginine, cysteine is also well known site of mono-ADP-ribosylation [31, 32]. Although mono-ART that modify b subunit of G proteins is characterized as arginine-specific [16], this specificity is not primarily
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referred to the modified amino acid residue but refers to the nearby binding areas [33]. Additionally, site-directed mutagenesis, loop grafting and co-crystallization [34, 35] studies revealed that the main specificity of mono-ADPtransferases is not at the target amino acid but rather on the surface areas near the modified amino acid residue. For example, exchanging the Glu/Gln residue, two residues upstream of the catalytic Glu, can convert an NAD?-hydrolase into an arginine-specific transferase (and vice versa) [36, 37] or an asparagine-specific ART into an arginine-specific ART [38]. Besides that, structures of ARTs co-crystallized with their target proteins uncovered a central role of one specific loop (ARTT loop) in target protein binding [39, 40] and experiments with mouse ART1 indicate there are sequences outside of the catalytic site that exert structural constraints, and that modulate the substrate specificity and catalytic activity [41]. Together, these experiments have not led to an easily recognizable substrate recognition motif, and at present the substrate specificity for a given ADP-ribosyltransferase can only be elucidated experimentally [33]. With present study, we completed a previous study [19] of mono-ADP-ribosylation of peptide, whose amino acid sequence corresponded to the third intracellular loop of GLP-1 receptor. Undoubtedly, this process occurs at applied conditions, the modified amino acid residues are Arg348 and Cys341 and in a viewpoint of G proteins b subunit it is referred as a competitive inhibition. Furthermore, Arg348 is modified predominantly, probably due to a greater similarity with native mono-ART substrate. In the light of the evolution, mono-ADP-ribosylation of Cys341 could evolve not so long ago as an alternative mechanism of receptor desensitization in case of Arg348 mutation. However, if this process also occurs under physiological conditions and if it possesses some regulatory role in signal transduction in vivo is rather speculative at this point, but with our findings possibility of aforementioned process in vivo was placed on a firm basis. In future, different biochemical approaches in vivo and in vitro [42–44] must be carefully used to elucidate the role of GLP-1 receptor regulation by mono-ART in normal and pathological physiology in mammals.
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