Trypsin cleavage of the β₁-adrenergic receptor

Abstract

β₁-Adrenergic receptors (β₁ARs) are the principal mediators of catecholamine action in cardiomyocytes. We previously showed that β₁ARs accumulate as both full-length and NH₂-terminally truncated species in cells, that maturational processing of full-length β₁ARs to an NH₂-terminally truncated form is attributable to O-glycan-regulated proteolytic cleavage of the β₁AR NH₂-terminus at R³¹ ↓ L³² by ADAM17, and that NH₂-terminally truncated β₁ARs remain signaling competent but they acquire a distinct signaling phenotype. NH₂-terminally truncated β₁ARs differ from full-length β₁ARs in their signaling bias to cAMP/PKA versus ERK pathways and only the NH₂-terminally truncated form of the β₁AR constitutively activates AKT and confers protection against doxorubicin-dependent apoptosis in cardiomyocytes. Since the R³¹ ↓ L³² sequence conforms to a trypsin consensus cleavage site, we used immunoblotting methods to test the hypothesis that β₁ARs are also cleaved at R³¹ ↓ L³² by trypsin (an enzyme typically used to isolate cardiomyocytes from the intact ventricle). We show that full-length β₁ARs are cleaved by trypsin and that trypsin cleaves the full-length β₁AR NH₂-terminus specifically at R³¹ ↓ L³² in CHO-Pro5 cells. Trypsin also cleaves β₁ARs in cardiomyocytes, but at a second site that results in the formation of ∼40-kDa NH₂-terminal and ∼30-kDa COOH-terminal fragments. The observation that cardiomyocyte β₁ARs are cleaved by trypsin (a mechanism that constitutes a heretofore-unrecognized mechanism that would influence β₁AR-signaling responses) suggests that studies that use standard trypsin-based procedures to isolate adult cardiomyocytes from the intact ventricle should be interpreted with caution.

NEW & NOTEWORTHY Current concepts regarding the molecular basis for β₁AR responses derive from literature predicated on the assumption that β₁ARs signal exclusively as full-length receptor proteins. However, we recently showed that β₁ARs accumulate as both full-length and NH₂-terminally truncated forms. This manuscript provides novel evidence that β₁-adrenergic receptors can be cleaved by trypsin and that cell surface β₁AR cleavage constitutes a heretofore unrecognized mechanism to alter catecholamine-dependent signaling responses.

INTRODUCTION

β₁-Adrenergic receptors (β₁ARs) are the principal mediators of catecholamine action in cardiomyocytes. We previously showed that the β₁AR extracellular NH₂-terminus is a target for posttranslational modifications that impact on signaling responses. Specifically, we showed that the β₁AR NH₂-terminus carries O-glycan modifications at Ser³⁷ and Ser⁴¹, that β₁ARs accumulate as both full-length and NH₂-terminally truncated species in cardiomyocytes and other cells types, and that maturational processing of the full-length β₁AR to an NH₂-terminally truncated species is attributable to O-glycan-regulated proteolytic cleavage of the β₁AR NH₂-terminus at R³¹ ↓ L³² by a disintegrin and metalloproteinase 17 [ADAM17 (1–3)]. Importantly, NH₂-terminal cleavage at this site has important functional implications. Although current concepts regarding the molecular basis for βAR responses derive from literature predicated on the assumption that β₁ARs signal exclusively as full-length receptor proteins, we showed that full-length and NH₂-terminally truncated forms of the β₁AR differ in their signaling bias to cAMP/PKA versus ERK pathways. Moreover, only the NH₂-terminally truncated form of the β₁AR couples to a Gi-dependent pathway that constitutively activates AKT and confers protection against doxorubicin-dependent apoptosis in cardiomyocytes (1–3). Our studies implicate the β₁AR extracellular NH₂-terminus as a heretofore unrecognized structural determinant of β₁AR activation. They expose a novel paradigm for the regulation of β₁AR signaling responses involving proteolytic cleavage of the extracellular NH₂-terminus.

The observation that the β₁AR is a target for limited NH₂-terminal proteolysis during maturational processing of the receptor protein raises the question of whether full-length β₁ARs on the cell surface can be cleaved by other proteases. This question seemed particularly relevant since sequence analysis reveals that the β₁AR NH₂-terminal R³¹ ↓ L³² cleavage site conforms to a trypsin consensus cleavage site (trypsin cleaves peptides COOH-terminal to lysine or arginine residues); we previously showed that the trypsin-based procedure used to isolate adult cardiomyocytes from the intact ventricle results in limited proteolysis of the β₁AR (4). This study tests the hypothesis that trypsin cleaves the β₁AR.

MATERIALS AND METHODS

Materials

Antibodies were from the following sources: rabbit polyclonal anti-β₁AR (ab3442, raised against residues 394–408 in human β₁-ARs) was from Abcam (Cambridge, MA). Studies from our laboratory validating the specificity of this antibody for the β₁AR are published previously (3). Mouse monoclonal anti-FLAG M2 antibody (Cat. No. F1804) was from Sigma Aldrich (St. Louis, MO). IRDye 800CW goat anti-rabbit IgG (Cat. No. 925–32211) and IRDye 680RD goat anti-mouse IgG (Cat. No. 925–68070) secondary antibodies were from LI-COR Biosciences (Lincoln, NE). Trypsin (Cat. No. T-161-25) was from Gold Bio (St. Louis, MO). Bovine lung aprotinin (Cat. No. A1153), benzamidine hydrochloride (Cat. No. 434760), and leupeptin (Cat. No. L2884) were from Sigma Aldrich. Phenylmethylsulfonyl fluoride (PMSF, Cat. No. 10837091001) was from Roche. All other chemicals were reagent grade.

Plasmids and Adenoviruses

The pcDNA3 human S⁴⁹R³⁸⁹-β₁AR harboring an NH₂-terminal Flag-tag was obtained from Addgene (Watertown, MA). The cleavage-resistant mutant construct β₁AR-31/52 (β₁AR-R31H/L32A/P52A/L53A) was generated using QuickChange II XL Site-Directed Mutagenesis Kits (Agilent Cat. No. 200522, Santa Clara, CA) according to the manufacturer’s instructions. Plasmids that drive expression of NH₂-terminally truncated forms of the human β₁AR (Δ2–31-β₁AR and Δ2–52-β₁AR) were kindly provided by Dr. Ulla E. Petäjä-Repo from the University of Oulu, Finland (5). Adenoviruses that drive expression of full-length, cleavage resistant, or NH₂-terminally truncated forms of the human β₁AR (Ad-WT-β₁AR, Ad-β₁AR-31/52, and Ad-Δ2–31-β₁AR) were prepared by Welgen, Inc. (Worcester, MA).

Chinese Hamster Ovary-Pro5 Cell Culture and Transfection

Chinese hamster ovary (CHO)-Pro5 cells (obtained from American Type Culture Collection, Cat. No. CRL-1781, Manassas, VA) were cultured in minimum essential medium Eagle-α modification (α-MEM), supplemented with 5% fetal bovine serum, penicillin (100 U/mL), streptomycin (100 µg/mL), and 100 mM glutamine. Transfections were performed with the jetOPTIMUS DNA Transfection Reagent (Polyplus Transfection, Cat. No. 101000006, Illkirch, France) according to the manufacturer’s instructions. The transfected cells were cultured for 24 h before harvest. For trypsin-inhibitor treatment, the cells were cultured in the presence of inhibitors for 24 h before harvest.

Cardiomyocyte Culture and Adenoviral Infections

Cardiomyocytes were isolated from the ventricles of 2-day-old Wistar rats (obtained from Charles River) by a trypsin dispersion procedure using differential attachment procedure to enrich for cardiomyocytes followed by irradiation as described previously (2). Methods to infect cardiomyocytes with adenoviruses that drive expression of full-length or NH₂-terminally truncated forms of human β₁AR (Ad-FL-β₁AR and Ad-Δ2–31-β₁AR) were previously published (2).

Immunoblotting

Immunoblotting with primary antibodies (anti-β1AR at 1:4,000 or anti-Flag at 1:1,000) and secondary antibodies (anti-rabbit or anti-mouse IgG, each at 1:5,000) was performed on cell extracts according to methods described previously (2) or the manufacturer’s instructions. All results were replicated in at least three experiments on separate culture preparations.

RESULTS

β₁AR Cleavage by Trypsin in CHO-Pro5 Cells

We previously used a mutagenesis strategy to show that the NH₂-terminally truncated form of the β₁AR is generated because of ADAM17-dependent cleavage of the full-length β₁AR at R³¹ ↓ L³² (1–3). We used a similar strategy (with the panel of constructs schematized in Fig. 1A) to determine whether R³¹ ↓ L³² also serves as a target for proteolytic cleavage by trypsin.

Figure 1.

Trypsin cleaves the FL-β₁AR at R³¹↓L³² in CHO-Pro5 cells. A: schematic of the NH₂-terminus of the NH₂-terminally Flag-tagged WT-β₁AR, the β₁AR mutant harboring single residue substitutions at the R³¹↓L³² cleavage site (and at a second putative cleavage site at P⁵²↓L⁵³), and the COOH-terminally Flag-tagged Δ2-31-β₁AR or Δ2-52-β₁AR truncation mutants used in our experiments. The downward arrow (↓) in the schematic denotes a cleavage site. B: CHO-Pro5 cells that express the WT-β₁AR, the cleavage resistant β₁AR-31/52 mutant, or Δ2-31-β₁AR or Δ2-52-β₁AR truncation mutants were incubated with the indicated concentration of trypsin for 10 min. Lysates were subjected to immunoblot analysis with an anti-β₁AR that recognizes a COOH-terminal epitope and anti-FLAG. The positions of the full-length WT-β₁AR and the cleavage resistant β₁AR-31/52 mutant are denoted by a filled black circle. The position of the NH₂-terminally truncated form of the WT-β₁AR is denoted by an unfilled circle. Positions of the Δ2-31-β₁AR or Δ2-52-β₁AR truncation mutants are denoted by filled green triangles. Results for trypsin-dependent changes in the abundance of the full-length form (filled circle) and NH₂-terminally truncated form (unfilled circle) of the WT-β₁AR (normalized to the abundance of the cognate protein prior to trypsin treatment) are quantified (means ± SE; n = 5 separate experiments). C: CHO-Pro5 cells that express the WT-β₁AR or the cleavage resistant β₁AR-31/52 were incubated for 10 min with 20 μg/mL trypsin following a 24 h pretreatment with trypsin inhibitor 1 (aprotinin/PMSF) or trypsin inhibitor 2 (benzamidine/leupeptin) as indicated. All results are representative of three to five separate experiments performed on separate culture preparations. D: schematic showing that trypsin cleavage of the WT-β₁AR is restricted to a single site on the NH₂-terminus at R³¹↓L³².

Figure 1B, left, shows that trypsin treatment leads to a dose-dependent decrease in the abundance of the full-length β₁AR species, and an increase in the NH₂-terminally truncated species, in CHO-Pro5 cell and that trypsin does not cleave a β₁AR-31/52 construct that harbors single residue substitutions that disrupt the trypsin consensus cleavage site at R³¹ ↓ L³² and a second predicted cleavage site at P⁵² ↓ L⁵³. We would note that the P⁵² ↓ L⁵³ cleavage site was identified in in vitro cleavage assays with short peptides based upon the β₁AR NH₂-terminal sequence as substrate and purified matrix metalloproteinase [MMP] or ADAM family enzymes (6), but its importance as a target for β₁AR cleavage in cells remains uncertain (1). Figure 1C shows that trypsin cleavage is prevented by inhibitors of trypsin activity.

Since trypsin cleaves peptides COOH-terminal to arginine or lysine residues and since all three β₁AR extracellular loops contain arginine residues, our experiments were designed to detect any trypsin cleavage events that would result in the accumulation of smaller β₁AR fragments. Figure 1B, left, shows that trypsin treatment does not lead to the accumulation of β₁AR NH₂- or COOH-terminal fragments in CHO-Pro5 cells, and Fig. 1B, right, shows that trypsin does not cleave NH₂-terminally truncated forms of the β₁AR. Δ2–31-β₁AR (a truncation mutant designed to mimic the NH₂-terminally truncated species formed as a result of full-length β₁AR cleavage at R³¹ ↓ L³²) or Δ2–52-β₁AR (a truncation mutant designed to mimic a species that would be formed as a result of β₁AR cleavage at a second putative cleavage site at P⁵² ↓ L⁵³) are not influenced by the trypsin treatment. This result emphasizes that trypsin cleavage of the β₁AR in CHO-Pro5 cells is confined to a single site at R³¹ ↓ L³² in the NH₂-terminus (as schematized in Fig. 1D); other potential cleavage sites on the extracellular surface of the receptor are not exposed, even when the NH₂-terminus (and all glycosylation sites) are removed from the receptor core.

β₁AR Cleavage by Trypsin in Cardiomyocytes

CHO-Pro5 cells provide a convenient, low-cost model for initial screens, but conclusions ultimately must be based on studies in the more physiologically relevant cardiomyocyte context. Therefore, the analysis was repeated in neonatal cardiomyocyte cultures. Figure 2A shows that trypsin cleaves the β₁AR in cardiomyocytes. However, in this case, trypsin treatment leads to a dose-dependent decrease in the abundance of both the full-length and the NH₂-terminally truncated forms of the β₁AR and it is accompanied by the accumulation of two β₁AR fragments; a ∼40-kDa FLAG-tagged NH₂-terminal fragment (detected by the anti-FLAG antibody) and a ∼30-kDa COOH-terminal fragment (detected by the anti-β₁AR antibody that recognizes a COOH-terminal epitope). The ∼40-kDa FLAG-tagged NH₂-terminal fragment must represent trypsin cleavage of the full-length β₁AR since a fragment derived from an NH₂-terminally truncated form of the β₁AR (either a species that exists at baseline in cells or one formed acutely because of trypsin cleavage of the full-length receptor at R³¹ ↓ L³²) would not be epitope-tagged. The ∼30-kDa COOH-terminal fragment represents cleavage of either full-length or NH₂-terminally truncated β₁AR species (see schematic in Fig. 2C). Of note, the size of the NH₂- and COOH-terminal fragments that accumulate in trypsin-treated cardiomyocytes is consistent with an intramolecular cleavage COOH-terminal at a pair of evolutionarily conserved arginine residues (adjacent to a disulfide bond-forming cysteine residue) at the tip of extracellular loop 2. The additional observation that trypsin cleaves the NH₂-terminally truncated Δ2–31-β₁AR mutant (Fig. 2B), generating a COOH-terminal fragment that comigrates with the COOH-terminal fragment formed because of trypsin cleavage of the WT-β₁AR (compare Fig. 2, A and B) provides unambiguous evidence that trypsin cleavage is not restricted to the NH₂-terminal site at R³¹ ↓ L³², but rather that trypsin cleaves the cardiomyocyte β₁AR at a second site tentatively mapped to extracellular loop 2. Moreover, the fact that trypsin treatment leads to the accumulation of a ∼40-kDa FLAG-tagged fragment (see Fig. 2A) provides strong evidence that this intramolecular cleavage does not require prior NH₂-terminal cleavage at R³¹ ↓ L³².

Figure 2.

Trypsin cleaves the FL-β₁AR in cardiomyocytes. Cardiomyocytes that express the NH₂-terminally FLAG-tagged WT-β₁AR (A) or the COOH-terminally tagged Δ2-31-β₁AR truncation mutant (B) were treated with the indicated concentrations of trypsin for 10 min and immunoblot analysis was performed with the anti- β₁AR antibody or anti-FLAG. Note: anti-FLAG detects the NH₂-terminal fragment generated because of trypsin cleavage of the NH₂-terminally FLAG-tagged WT-β₁AR (A), but the COOH-terminal fragment generated because of trypsin cleavage of the COOH-terminally FLAG-tagged Δ2-31-β₁AR truncation mutant (B). A representative experiment is illustrated on top with results for trypsin-dependent changes in the abundance of full-length and NH₂-terminally truncated forms of the WT-β₁AR (normalized to protein abundance prior to trypsin treatment) quantified at the bottom (means ± SE; n = 4). C: schematic showing that trypsin cleaves the full-length WT-β₁AR and the Δ2-31-β₁AR truncation mutant at an intramolecular site generating a ∼40-kDa NH₂-terminal fragment from the full-length WT-β₁AR (depicted in red), a somewhat smaller NH₂-terminal fragment from the Δ2-31-β₁AR truncation (depicted in pink, since it is not tagged and would not be detected) and a ∼30-kDa COOH-terminal fragment from both the full-length WT-β₁AR and the Δ2-31-β₁AR truncation mutant (depicted in red). Although trypsin is predicted to also cleave the full-length WT-β₁AR at R³¹↓L³², this species (depicted in purple) cannot be identified by this method since it would comigrate with the NH₂-terminally truncated β₁AR species present at baseline in cardiomyocytes (and its appearance might be transient, since it presumably would be further cleaved to the smaller NH₂- and COOH-terminal fragments).

DISCUSSION

This study provides novel evidence that the β₁AR is cleaved by trypsin, that trypsin cleaves the full-length β₁AR at R³¹ ↓ L³² (the site that serves as a target for ADAM17-dependent cleavage of the β₁AR NH₂-terminus during maturational processing of the receptor) in CHO-Pro5 cells, and that trypsin cleaves both full-length and NH₂-terminally truncated β₁ARs at a second site tentatively mapped to extracellular loop 2 in cardiomyocytes (see schematics in Figs. 1D and 2C). A mechanism that might fully explain this cell-specific difference in β₁AR cleavage by trypsin is not obvious. It is tempting to speculate that these distinct cleavage patterns could be due (at least in part) to differences in the trafficking pattern and/or subcellular compartmentation of full-length versus NH₂-terminally truncated β₁ARs in CHO-Pro5 cells versus cardiomyocytes. According to this formulation, trypsin’s actions would be specific for the full-length β₁AR in CHO-Pro5 cells if this was the only β₁AR species that traffics to the cell surface (i.e., if NH₂-terminally truncated β₁ARs are trapped in intracellular compartments and are protected), whereas trypsin would cleave both full-length and NH₂-terminally truncated β₁AR species in cardiomyocytes if they are both expressed on the cell surface. However, this mechanism would not adequately explain the additional observation that trypsin cleaves the full-length β₁AR at a single NH₂-terminal site at R³¹ ↓ L³² in CHO-Pro5 cells, whereas it cleaves both full-length and NH₂-terminally truncated β₁ARs at a second intramolecular site in cardiomyocytes. Given the emerging evidence that the NH₂-termini of certain G protein-coupled receptors play a structural role to pack against the receptor’s extracellular surface [and that glycan modifications can influence this type of interaction (7)], it is interesting to speculate that cell-specific differences in glycan modifications might stabilize an NH₂-terminal conformation that either shields or exposes an extracellular loop cleavage site and thereby influences proteolytic cleavage. Alternatively, cell-specific differences in trypsin cleavage could be due to differences in receptor interactions with binding partners that dock to the β₁AR intracellular surface and impose structural constraints on the receptor core that impact on the conformation of extracellular loop 2 and cleavage site exposure. These mechanisms are not mutually exclusive and are the focus of ongoing studies in our laboratory.

The two distinct β₁AR cleavage patterns identified in our study are predicted to have very different functional implications. The trypsin-dependent cleavage mechanism identified in CHO-Pro5 cells, which is restricted to the full-length β₁AR and is at a single NH₂-terminal site at R³¹ ↓ L³², effectively converts cell surface full-length β₁ARs to the NH₂-terminally truncated form. We previously used a heterologous overexpression strategy to show that the NH₂-terminally truncated form of the β₁ARs differs from the full-length β₁AR in its signaling bias to cAMP/PKA versus ERK pathways and only the NH₂-terminally truncated form of the β₁AR constitutively activates a Gi-AKT pathway that confers protection against doxorubicin-dependent apoptosis (2, 3). Although it is tempting to speculate that NH₂-terminally truncated β₁ARs generated on the cell surface as a result of trypsin’s actions will phenocopy this activity profile, we would caution against any premature conclusions since they would be predicated on the implicit assumption that the NH₂-terminally truncated form of the receptor generated as a result of cleavage of full-length β₁ARs on the cell surface by inflammatory proteases, the NH₂-terminally truncated receptor species formed during maturational processing of full-length β₁ARs in cells, and a heterologously overexpressed Δ2–31-β₁AR transgene couple to identical signaling pathways and cellular responses. In theory, a protease generated NH₂-terminally truncated β₁AR might be confined to the cell surface, whereas the NH₂-terminally truncated endogenous receptor species formed during maturational processing of full-length β₁ARs (or a Δ2–31-β₁AR transgene) might localize or traffic to a distinct intracellular compartment. These distinct localization patterns could facilitate or restrict interactions with binding partners and impact on the β₁AR signaling phenotype.

The functional implications of the trypsin-dependent intramolecular cleavage mechanism identified in neonatal cardiomyocyte cultures would be quite different. Here, cleavage at an extracellular loop 2 site is predicted to disrupt the transmembrane ligand-binding pocket and lead to a defect in β₁AR responses (8, 9). Although studies to determine whether the factors that facilitate an intramolecular β₁AR disabling cleavage in cultured neonatal cardiomyocytes also exist in adult cardiomyocytes (or cardiomyocytes that have induced hypertrophy) are critical, both cleavage patterns identified in our studies (cleavage at R³¹ ↓ L³² in the NH₂-terminus or at an intramolecular extracellular loop 2 site) would have important functional implications. These results suggest caution in the interpretation of studies that interrogate catecholamine responses in adult cardiomyocytes acutely isolated from the intact ventricle using standard trypsin-based protocols. In this context, it is worth noting that trypsin-based protocols also are used in the preparation of neonatal cardiomyocyte cultures, but this would not confound the interpretation of studies on neonatal cardiomyocyte culture preparations since such studies typically are performed 4–5 days following cell isolation; endogenous β₁AR receptors are newly synthesized during this culture interval and any β₁AR transgenes introduced into cells after cell isolation were never been exposed to the trypsin treatment. Finally, we would note that we recognize that our studies optimally would have tracked trypsin cleavage of the endogenous β₁AR in cardiomyocytes. Although this approach was included in our previous publications, our recent attempts to track endogenous β₁ARs in cardiomyocytes have proved unsuccessful since the Santa Cruz anti-β₁AR previously validated and used for this purpose is no longer available (1, 3) and exhaustive screens of all available anti-β₁AR antibodies have failed to identify a reagent with the requisite specificity/sensitivity to detect endogenous β₁ARs in cardiomyocytes. However, our previous observation that the expression patterns (the ratio of full-length vs. NH₂-terminal truncated species) and regulation of endogenous and heterologously overexpressed β₁ARs in cardiomyocytes is similar suggests that receptor overexpression is not a significant confounding factor in our studies.

Although the in vivo physiological importance of β₁AR cleavage by trypsin (a digestive enzyme in the gastrointestinal tract) in the heart is somewhat dubious, our results raise the intriguing possibility that the β₁AR NH₂-terminus might be a target for cleavage by other more physiologically relevant serine proteases. For example, mast cell tryptase (an enzyme with a consensus cleavage motif similar to that described for trypsin) is released by activated mast cells at sites of myocardial injury (10). Evidence that tryptase acts like trypsin to cleave the β₁AR NH₂-terminus would have important pathophysiological implications given evidence that mast cells play a functionally important role to preserve postischemic cardiac reserve (11). A previous study attributed this to tryptase cleavage/activation of cardiomyocyte PAR-2 (11) [a Gi-coupled GPCR that confers protection against ischemia-reperfusion injury (12, 13)], but tryptase cleavage of the β₁AR (a mechanism that would lead to the accumulation of a cardioprotective NH₂-terminally truncated β₁AR species) could constitute an additional novel cardioprotective mechanism.

GRANTS

This work was supported by the National Heart, Lung, and Blood Institute Grant HL138468 (to S.F.S).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

S.F.S. conceived and designed research; J.Z. performed experiments; S.F.S. and J.Z. analyzed data; S.F.S. and J.Z. interpreted results of experiments; S.F.S. prepared figures; S.F.S. drafted manuscript; S.F.S. and J.Z. edited and revised manuscript; S.F.S. and J.Z. approved final version of manuscript.