©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Ligand-dependent G Protein Coupling Function of Amyloid Transmembrane Precursor (*)

(Received for publication, November 11, 1994; and in revised form, January 4, 1995)

Takashi Okamoto (1)(§) Shizu Takeda (1) Yoshitake Murayama (2) Etsuro Ogata (3) Ikuo Nishimoto (1)(§)

From the  (1)Cardiovascular Research Center, Massachusetts General Hospital and Department of Medicine, Harvard Medical School, Charlestown, Massachusetts 02129, the (2)Fourth Department of Internal Medicine, Tokyo University School of Medicine, 3-28-6 Mejirodai, Bunkyo-ku, Tokyo 112, Japan, and the (3)Cancer Research Institute, 1-37-1 Kami-Ikebukuro, Toshima-ku, Tokyo 170, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Amyloid precursor protein (APP), a transmembrane precursor of beta-amyloid, possesses a function whereby it associates with G(o) through its cytoplasmic His-Lys. Here we demonstrate that APP has a receptor function. In phospholipid vesicles consisting of baculovirally made APP and brain trimeric G(o), 22C11, a monoclonal antibody against the extracellular domain of APP, increased GTPS binding and the turnover number of GTPase of G(o) without affecting its intrinsic GTPase activity. This effect of 22C11 was specific among various antibodies and was observed neither in G(o) vesicles nor in APP/G vesicles. In APP/G(o) vesicles, synthetic APP, the epitope of 22C11, competitively antagonized the action of 22C11. Monoclonal antibody against APP, the G(o) binding domain of APP, specifically blocked 22C11-dependent activation of G(o). Therefore, APP has a potential receptor function whereby it specifically activates G(o) in a ligand-dependent and ligand-specific manner.


INTRODUCTION

Alzheimer's disease (AD) (^1)is a progressive neurodegenerative disorder causing dementia (Katzman, 1986). There are two pathological hallmarks of depositions: extracellular amyloid plaques and intracellular paired helical filaments. The former structures consist of beta-amyloid. In AD, beta-amyloid is inferred to deposit by being cleaved off from a transmembrane precursor, termed APP. After the identification of APP by Kang et al.(1987), which consists of 695 residues, at least 10 isoforms of APP have been identified as a result of alternative splicing of a single gene (Sandbrink et al., 1994). APP is preferentially expressed in the brain.

In patients with dominantly inherited AD (FAD), point mutations have been discovered in the transmembrane domain of APP. The Val of APP is converted to Ile (Goate et al., 1991; Naruse et al., 1991; Yoshioka et al., 1991), Phe (Murrell et al., 1991), or Gly (Chartier-Harlin et al., 1991). They co-segregate with the disease phenotype (Karlinsky et al., 1992), indicating that structural alteration of APP is a cause of AD. APP structurally resembles a cell surface receptor possessing a single transmembrane region (Dyrks et al., 1988) and is actually localized in cellular membranes (Schubert et al., 1991). Although these findings suggest that APP functions as a cell surface receptor, this theory was difficult to prove because of the lack of information about both the extracellular ligand and the intracellular signal of APP. We have, however, found that His-Lys of APP selectively activates G(o) and that intact APP forms a complex with the heterotrimeric G protein G(o) through this domain in a GTPS-inhibitable manner (Nishimoto et al., 1993). Given the established role of heterotrimeric G proteins in receptor signaling, these observations provide the first evidence that APP encodes a signaling receptor.

Based on these findings, we demonstrate here by reconstituting purified proteins into phospholipid vesicles that APP mimics a functional G(o)-coupled receptor regulated by a ligand. Since the natural ligand for APP is unknown, we have used 22C11 as a potential agonist. 22C11 is a mAb raised against E. coli-made APP (Weidemann et al., 1989), whose epitope region has been assigned to APP in the ectoplasmic Cys-containing domain (Hilbich et al., 1993). Although the recognition potency is not very high, antibody absorption (Milward et al., 1992) and immunoprecipitation (Nishimoto et al., 1993) indicate that this mAb does recognize a native form of APP. This study, in which we conclude that APP serves as a normal G(o)-coupled receptor, provides a novel insight into both physiological and pathological roles of APP.


EXPERIMENTAL PROCEDURES

Baculovirus encoding normal APP gene has been described previously (Nishimoto et al., 1993). APP was purified from the membranes of Sf21 insect cells infected with this baculovirus using two-step chromatography, as described (Moir et al., 1992) with slight modification. Briefly, after washing insect cells with phosphate-buffered saline and centrifuging them, the precipitates were solubilized with homogenization buffer consisting of 10 mM Tris/HCl (pH 7.5), 100 mM NaCl, 5 mM EDTA, 1% (v/v) Triton X-100, 2 mM phenylmethylsulfonyl fluoride, 0.2 TIU/ml aprotinin, and 2 mM leupeptin with mixing every 5 min for 1 h at 4 °C. The lysate was centrifuged at 3000 rpm for 1 h at 4 °C. After adjusting the NaCl and EDTA concentrations to 350 mM and 5 mM, the supernatant was loaded onto a Q-Sepharose column. The column was then washed with 50 mM Tris/HCl (pH 7.4), 5 mM EDTA, 350 mM NaCl, and 1% Triton X-100. Bound proteins were eluted with 50 mM Tris/HCl (pH 7.4), 5 mM EDTA, and 1 M NaCl. The peak protein fractions from the Q-Sepharose column were pooled and applied to a G-25 Sepharose desalting column, through which the buffer containing 50 mM Tris/HCl (pH 7.4), 5 mM EDTA, and 175 mM NaCl was prompted. The protein fraction from the G-25 column was applied to a heparin-Sepharose column. After the column was washed with homogenization buffer, bound protein was eluted with 50 mM Tris/HCl (pH 7.4), 5 mM EDTA, and 650 mM NaCl. Trimeric G(o) (Katada et al., 1986) and G (Katada et al., 1987) purified from bovine brain to homogeneity were provided by Dr. T. Katada, stored in 20 mM Hepes/NaOH (pH 7.4), 0.1 mM EDTA, and 0.7% CHAPS, and diluted geq10-fold for reconstitution. APP and trimeric G(o) were reconstituted using the gel filtration method Nishimoto et al., 1989). During reconstitution, premature activation of G(o) was strictly prohibited by cooling vesicles at 4 °C as well as by removing Mg from the solution. Reconstitution of APP with G was done in a similar manner. Like vesicles consisting of APP and G(o), these vesicles only consisted of APP and G (data not shown). 22C11 and anti-beta-adrenergic receptor antibody were from Boehringer Mannheim and Oncogene Science, respectively. AC-1 was kindly provided by Dr. K. Yoshikawa (Yoshikawa et al., 1992). 4G5 and 1C1 were described (Nishimoto et al., 1993). The epitope peptide APP for 22C11 (KEGILQYCQEVYPELQ) was synthesized and purified to >97% purity.

Vesicles were incubated with stimulation at 37 °C, and either GTPase activity or GTPS binding activity was measured. The turnover number of GTPase activity was assayed as described (Okamoto et al., 1991). Unless otherwise specified, the turnover number of GTPase activity was measured by incubating G proteins with 100 nM [-P]GTP in the presence of 20 µM Mg for 20 min. GTPS binding to G proteins was assayed in the presence of 20 µM Mg, and the rate constant k was calculated as described (Okamoto and Nishimoto, 1992). Intrinsic GTPase activity was measured as described (Strittmatter et al., 1991). All assays were performed independently at least three times, usually six times, with similar results.


RESULTS AND DISCUSSION

Intact APP was purified from insect cell homogenate infected with the recombinant baculovirus encoding APP cDNA and was reconstituted with purified trimeric G(o) into phospholipid vesicles. Silver staining revealed that APP and G(o) were virtually the only proteins that were contained in the reconstituted vesicles (Fig. 1A). Since soluble forms of APP are possible contaminants, the membrane precipitate of infected insect cells was used as a starting material to ensure the purification of full-length APP. The preparation of APP used for reconstitution only contained a 130-kDa protein. This protein was immunoreactive with anti-APP mAb 22C11 (data not shown), verifying that the target of 22C11 in reconstituted vesicles is only APP. It has been reported that insect cells infected with baculovirus encoding APP cDNA secrete a truncated APP lacking the C terminus (Knops et al., 1991). However, the purified APP was a full-length APP, as it reacted with both the extreme C-terminal antibody AC-1 and the ectoplasmic domain antibody 22C11 in immunoblot analysis (data not shown).


Figure 1: Anti-APP antibody 22C11 activates G(o) in APP/G(o) vesicles. A, silver staining of APP/G(o) vesicles. APP (40 pmol) was reconstituted with trimeric form of G(o) (20 pmol) into phospholipid vesicles. The reconstituted vesicles were applied to SDS-polyacrylamide gel electrophoresis and silver-stained. B, the APP/G(o) vesicles were incubated with 5 µg/ml 22C11 () or vehicle () for the indicated periods, and the turnover number of GTPase activity of G(o) was measured as assessed with radioactive phosphate released from [-P]GTP. All values represent means ± S.E. of at least three independent experiments. C, the APP/G(o) vesicles were incubated with 5 µg/ml 22C11 () or vehicle () for the indicated periods in the presence of [-S]GTPS, and GTPS binding to G(o) was measured, as described previously (Okamoto and Nishimoto, 1992). The total amount of G(o) in the vesicles was determined after reconstitution, based on the measurement of maximal GTPS binding in the presence of 10 mM Mg under the same condition, which yielded the consistent results with the assay reported (Northup et al., 1982). D, The APP/G(o) vesicles (rightpanel) or the vesicles reconstituted with trimeric G(o) alone (leftpanel) were incubated with or without several immunoglobulins (each 5 µg/ml), and the turnover number of GTPase activity was measured. In G(o) vesicles, trimeric G(o) (20 pmol) was similarly reconstituted into phospholipid vesicles.



When the APP/G(o) vesicles were incubated with 5 µg/ml 22C11, the turnover number of GTPase activity of G(o) was stimulated to 200-250% of the basal G(o) activity of the vesicles incubated without 22C11 (Fig. 1B). The rate of GTPS binding to G(o) was also promoted to 250% of the basal binding rate by the same concentration of 22C11 (Fig. 1C). In vesicles reconstituted with trimeric G(o) alone, little stimulation by 22C11 was observed (Fig. 1D), suggesting that the mediator of 22C11 is APP. The action of 22C11 was immunoglobulin-specific (Fig. 1D). None of the nonspecific IgG, 4G5, 1C1, or anti-beta-adrenergic receptor antibody stimulated G(o) in the same vesicles that allowed 150% stimulation of G(o) by 22C11. 4G5 and 1C1 are antibodies against two distinct cytoplasmic domains of APP. A slight but detectable difference in the basal G(o) activities between G(o) vesicles and APP/G(o) vesicles provides additional evidence that APP behaves like a G(o)-linked receptor, as there could be a basally active fraction in G-coupled receptors (Samama et al., 1993).

Coupling of APP seemed to be selective to G(o). When trimeric G was similarly reconstituted with APP and stimulated by 22C11, no significant stimulation of G was observed (Fig. 2A). This is highly consistent with our previous study, which detected specific activation of G(o) by APP as well as specific association of intact APP with G(o) (Nishimoto et al., 1993).


Figure 2: Characterization of 22C11-dependent G(o) activation by APP. A, effect of 22C11 in APP/G vesicles. APP (40 pmol) was reconstituted with trimeric G (20 pmol) into phospholipid vesicles. The turnover number of GTPase activity of G was measured by incubating the vesicles with or without 5 µg/ml 22C11. All values represent means ± S.E. of at least three independent experiments, unless otherwise specified. B, dose-response effect of 22C11 and competitive inhibition by the 22C11 epitope peptide. The APP/G(o) vesicles used in Fig. 1were incubated with increasing concentrations of 22C11 that had been pretreated with water (hatchedbar) or the 22C11 epitope peptide (APP, finally 1 mM) (blackbar), and the turnover number of GTPase activity was measured. Treatment of 22C11 with the epitope peptide or water was carried out for 40 min at room temperature. The values corresponding to 100% and 0% stimulations are 0.39 and 0.17 (min), respectively. C, effect of 4G5 on 22C11-induced G(o) activation by APP. The APP/G(o) vesicles were incubated with 5 µg/ml 22C11 in the presence of increasing concentrations of 4G5 (black bar) or 1C1 (hatchedbar), and the turnover number of GTPase activity was measured. 4G5 or 1C1 had no effect on the G(o) activity without 22C11. D, effect of 22C11 on the intrinsic GTPase activity of G(o) in APP/G(o) vesicles. The APP/G(o) vesicles were incubated with [-P]GTP in the Mg-free buffer consisting of 20 mM Hepes/NaOH (pH 7.4) and 0.1 mM EDTA at 20 °C for 16 min. Reaction was initiated by adding 1.3 mM MgSO(4) and 20 µM GTP with () or without () 5 µg/ml 22C11 at 37 °C, and was terminated with ice-cold charcoal after indicated periods of incubation. These experiments were done three times independently and showed similar results. This figure shows a representative result. E and F, Mg dependence of the 22C11 action on the turnover number of GTPase activity (E) or GTPS binding activity (F) of APP/G(o) vesicles. The APP/G(o) vesicles were incubated with [-P]GTP for 10 min (for GTPase) or with [-S]GTPS for 2 min (for GTPS binding) at various Mg concentrations in the absence or presence of 5 µg/ml 22C11. The concentration of free Mg was determined according to Iyengar and Birnbaumer(1982).



To verify the mediation of APP in the action of 22C11, we examined the interfering effect of APP, the epitope peptide for 22C11. As shown in Fig. 2B, 22C11 dose-dependently promoted the turnover number of GTPase activity of G(o) in APP/G(o) vesicles. When premixed with 22C11 for 40 min at room temperature, APP shifted the dose-response curve to the right with the potency of 22C11 being attenuated by severalfold and its efficacy being unaltered (Fig. 2B). This shows that APP competitively antagonizes the action of 22C11 in this system, being consistent with the fact that APP is the epitope for 22C11. The requirement for high concentrations of APP may be attributed to the low affinity with which the synthetic short epitope binds to the antibody relative to the affinity of the entire protein as well as to the short preincubation period. This provides strong evidence that 22C11 acts on APP to stimulate G(o) in APP/G(o) vesicles.

4G5, mAb against APP, had no effect in APP/G(o) vesicles by itself (Fig. 1D). Even at 200 µg/ml, neither 4G5 nor 1C1 affected the G(o) activity in these vesicles (data not shown). However, when APP/G(o) vesicles were incubated with 4G5 in addition to 22C11, the 22C11 effect on G(o) activity was inhibited (Fig. 2C). The inhibition was dose-dependent for 4G5 and nearly complete at 200 µg/ml. Whereas the effective concentration of 4G5 was high, 1C1, a mAb that recognizes APP (the 19-residue region adjacent to the 20-residue epitope for 4G5), failed to inhibit activity. These data offer excellent support to the idea that APP activates G(o) through His-Lys, which is a demonstrated G(o) binding and activating domain (Nishimoto et al., 1993).

In an effort to further characterize the mechanism for G(o) activation by 22C11 in APP/G(o) vesicles, we measured intrinsic GTPase of G(o) (Fig. 2D). Although steady state GTPase activity, which represents the turnover number of the G protein activation cycle, was promoted by 22C11, intrinsic GTPase activity was not affected by this antibody. This indicates that 22C11 stimulates GDP/GTP exchange of G(o) without altering its intrinsic GTP hydrolysis in APP/G(o) vesicles. Ligand stimulation of APP thus causes G(o) activation in a manner highly similar to that of receptors.

We also measured the Mg dependence of the 22C11 effect on G(o) activity in APP/G(o) vesicles (Fig. 2, E and F). Both assays of GTPS binding rate, which was assessed with the rate constant k, and of the turnover number of GTPase activity revealed that 22C11 most effectively stimulated G(o) over a wide range of Mg concentrations between 10 µM and 1 mM, showing the close similarity of the action of APP to that of conventional G-coupled receptors.

APP has been shown to complex with G(o) in a receptor-like manner, suggesting that it encodes a potential G(o)-coupled receptor (Nishimoto et al., 1993). The first significance of the present work is to demonstrate the ligand-dependent function in APP, which provides direct evidence of a receptor-like function of APP. Here it is shown that anti-APP mAb 22C11 specifically activates G(o) in APP/G(o) vesicles with the absolute requirement of APP for this effect of 22C11. Although 22C11 does not have high potency to interact with intact APP, as shown in Fig. 2B, this mAb does recognize a native form of APP, as has been shown by immunoprecipitation (Nishimoto et al., 1993) and antibody absorption (Milward et al., 1992). Inhibition of the 22C11 action by APP provides additional evidence that this antibody recognizes APP in APP/G(o) vesicles. It is also likely that this mAb, which belongs to divalent IgG1, activates the function of APP by facilitating its dimerization, as has been the mechanism whereby ligands turn on the functions of all the single-spanning receptors that have been investigated (Ullrich and Schlessinger, 1990; Fuh et al., 1992; Watowich et al., 1994; Spencer et al., 1993). The mode of G(o) activation by APP characterized here is highly similar to that of receptor-stimulated G protein activation. The function of APP is likely mediated by the His-Lys region. The finding that APP could not activate G in response to 22C11 clearly shows the action specificity of APP for the recognition of G proteins. This G protein specificity provides additional evidence that APP couples to G(o) through a specific mechanism.

The natural ligand for APP has not been established. However, the function of this protein is studied here through an activating mAb. Such a strategy has been successfully used in the anti-CD3 antibody/T cell receptor system (Imboden and Stobo, 1985), the Fas antigen system (Suda et al., 1993), and others. There are many precedent receptors in which antibodies mimic the function of native ligands. Given the fact that activation of APP/G(o) coupling by the agonistic antibody is virtually identical to the known receptor/G protein coupling by receptor ligands, it is highly likely that natural ligands also exist for APP. Several proteins such as transforming growth factor-beta (Bodmer et al., 1990) bind to APP. Our system is suitable for the search of the physiological APP ligands, which should not only bind to APP but also activate its function.

The present study points to a potential role for G(o) as the mediator of normal function of APP. There is growing evidence suggesting that APP is normally involved in synaptic contact (Schubert et al., 1991) and cell adhesion (Ueda et al., 1989; Chen and Yankner, 1991; Mönning et al., 1992). Multiple reports also indicate that G(o) is involved in signaling of neuronal adhesion (Schuch et al., 1989; Doherty et al., 1991), neurite outgrowth (Strittmatter et al., 1994), and growth cone regulation (Strittmatter et al., 1990). Our conclusion that APP has functional properties of a cell surface receptor coupled to G(o) is in excellent agreement with these observations.

The mediation of APP signals by G(o) also suggests that G(o) could contribute to the pathophysiology of AD. Indeed, the tissue distribution of G(o) in the brain corresponds well to the areas severely afflicted by AD. Thus, it is very important to examine whether the APP/G(o) coupling is abnormal in AD-derived tissues or in FAD-linked mutants of APP. Based on the extreme similarity of the point mutations found in the FAD-linked APPs to the oncogenic Neu mutation of c-ErbB2, a receptor tyrosine kinase (Bargmann et al., 1986), we assume that G(o) is constitutively activated by these APP mutants linked to FAD. There are multiple lines of evidence suggesting that G proteins, which could be G(o), are activated in AD tissues (Smith et al., 1989; Flynn et al., 1991; Gibson and Toral-Barza, 1992; Ito et al., 1994; Cowburn et al., 1992; Cowburn et al., 1993; Huang and Gibson, 1993). This study should therefore provide the basis for both physiological and pathological functions of APP.

There are many isoforms of APP except APP (Sandbrink et al., 1994). Regarding the novel function that this study has identified, difference or identity should be clarified between APP and other isoforms. Some of them have the extracellular domain identical to that of APP with a truncated cytoplasmic domain and could thereby work as a dominant-negative receptor for APP. Conversely, both APP and APP, which have the cytoplasmic domain identical to that of APP with different extracellular domains, could interfere the signaling function of APP inside the cells. Cells may respond to the putative APP ligand as a result from the whole body of molecular functions of these isoforms.


FOOTNOTES

*
This work was supported in part by Bristol-Myers Squibb and by grants from the Ministry of Education, Science, and Culture of Japan, Chugai, and Hoechst. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Cardiovascular Research Center, Massachusetts General Hospital and Dept. of Medicine, Harvard Medical School, 149 13th St., Charlestown, MA 02129. Tel.: 617-726-4348; Fax: 617-726-5806; nishimoto{at}helix.mgh.harvard.edu.

(^1)
The abbreviations used are: AD, Alzheimer's disease; APP, amyloid precursor protein; G protein, guanine nucleotide-binding protein with trimeric composition; FAD, familial Alzheimer's disease; mAb, monoclonal antibody; GTPS, guanosine-5`-O-(3-thiotriphosphate); CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.


ACKNOWLEDGEMENTS

We thank T. B. Kinane for critical reading of this manuscript and advice, A. Bush and R. E. Tanzi for the APP preparation used in the experiments preliminary to the experiments here, T. Katada for G(o) and G, K. Yoshikawa for AC-1, Y. Tamai for support, U. Giambarella and D. Wylie for expert technical assistance, and K. Yonezawa and M. C. Fishman for critical reading of this manuscript. We are also indebted to E. J. Neer, E. Haber, A. H. Tashjian, J. H. Growdon, B. A. Yankner, H. Potter, and G. D. Fischbach for indispensable discussion.


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