©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Mannose 6-Phosphate/Insulin-like Growth Factor II Receptor Fails to Interact with G-proteins
ANALYSIS OF MUTANT CYTOPLASMIC RECEPTOR DOMAINS (*)

(Received for publication, July 25, 1994; and in revised form, September 19, 1994)

Christian Körner (1) Bernd Nürnberg (2) Martina Uhde (2) Thomas Braulke (1)(§)

From the  (1)Institut für Biochemie II, Georg-August-Universität Göttingen, Gosslerstr. 12d, D-37073 Göttingen, Federal Republic of Germany and the (2)Institut für Pharmakologie, Freie Universität Berlin, Thielallee 67-73, D-14195 Berlin, Federal Republic of Germany

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The binding of insulin-like growth factor II (IGF II) to the mannose 6-phosphate (M6P)/IGF II receptor has previously been reported to induce the activation of trimeric G proteins by functional coupling to a 14-amino acid region within the cytoplasmic receptor domain (Nishimoto, I., Murayama, Y., Katada, T., Ui, M., and Ogata, E.(1989) J. Biol. Chem. 264, 14029-14038). In the present study, we examined further the potential functional coupling of G-proteins with the human M6P/IGF II receptor and mutant receptors lacking the proposed G-protein activator sequence. IGF II treatment of mouse L-cells expressing either wild type or mutant M6P/IGF II receptors failed to attenuate the pertussis toxin-catalyzed modification of a 40-kDa protein or enhance GTPase activity. In broken L-cell membranes expressing wild type or mutant M6P/IGF II receptors, 30 nM IGF II also failed to affect the pertussis toxin substrate activity. By using phospholipid vesicles reconstituted with human wild type or mutant M6P/IGF II receptors and pertussis toxin-sensitive G-proteins, no stimulation of GTPS binding to or GTPase activity of G, G, or G(i)/G(o) mixtures were observed in response to 1 µM IGF II. Furthermore, in vesicles containing purified wild type M6P/IGF II receptors and monomeric Galpha or Galpha and beta dimers no effects of IGF II on GTPS binding could be detected. However, when vesicles reconstituted with M6P/IGF II receptors and G proteins were incubated with 100 µM mastoparan GTPS binding was stimulated and GTPase activity was increased significantly. These results indicate that the human M6P/IGF II receptor neither interacts with G-proteins in mouse L-cell membranes nor is coupled to G proteins in phospholipid vesicles. This study suggests strongly that the M6P/IGF II receptor does not function in transmembrane signaling in response to IGF II.


INTRODUCTION

The 300-kDa mannose 6-phosphate (M6P) (^1)receptor medi-ates the transport of newly sythesized soluble acid hydrolases from the Golgi to an endosomal/prelysosomal compartment where dissociation of the ligands occurs. The released acid hydrolases are delivered to lysosomes while the receptors recycle back to the Golgi or move to the plasma membrane. About 10% of the 300-kDa M6P receptors are localized at the cell surface where they function in binding and internalization of exogenous lysosomal enzymes and the nonglycosylated insulin-like growth factor II (IGF II) (see Refs. 1 and 2 for review). Both classes of receptor ligand bind to distinct sites, and the receptor can bind lysosomal enzymes and IGF II simultaneously (3, 4, 5) .

IGF II is functionally and structurally related to IGF I, and both factors have been shown to act in an autocrine/paracrine fashion regulating growth and differentiation(6, 7, 8) . The use of various antibodies against IGF I or M6P/IGF II receptors(9, 10) , mutants of IGF II with reduced affinities for the M6P/IGF II receptor (11, 12) and targeted mutagenesis in mice (13) indicate that most of the metabolic and mitogenic effects of IGF II are mediated by the IGF I receptor. The IGF I receptor is composed of an extracellular ligand-binding alpha subunit and an intracellular beta subunit that contains an intrinsic ligand-activated tyrosine kinase. However, there are reports suggesting that certain IGF II responses are mediated by its binding to the monomeric M6P/IGF II receptor, which lacks tyrosine kinase activity in its cytoplasmic domain.

IGF II stimulates the Na/H exchange and the production of inositol trisphosphate and diacylglycerol in basolateral membranes of renal proximal tubular cells(14, 15) . Other studies showed that IGF II promotes amino acid uptake in myoblasts (16) and DNA synthesis and Ca influx in primed-competent BALB/c 3T3 cells(17) . The involvement of G-proteins in the latter processes was suggested, since the effects of IGF II were completely abolished by pretreatment of cells with pertussis toxin. These data were confirmed by the demonstration of IGF II-induced activation of isolated G proteins via functional coupling to purified M6P/IGF II receptors in phospholipid vesicles and cell membranes(18, 19) . Furthermore, the IGF II-induced activation of G(i) proteins could be inhibited by M6P and M6P-containing lysosomal enzymes(19) .

Because of structural similarities with mastoparan, a wasp venom peptide activating G(o) and G(i) proteins(20) , Nishimoto and co-workers (21) identified a region corresponding to residues 123-136 of the cytoplasmic domain of the human M6P/IGF II receptor, which they proposed may play a major role in the receptor-activating function on G(i) proteins. In contrast, the large family of known G-protein-coupled receptors is characterized by a common sequence pattern with seven hydrophobic transmembrane helices resulting in the same basic three-dimensional structure (for review see (22) ). Binding of ligands to those receptors stimulates the activation of trimeric G-proteins by catalyzing a GDP/GTP exchange, which initiates either intracellular second messenger formation or regulation of ion channel function(23) .

In the present study we reevaluate the previously suggested participation of M6P/IGF II receptors in a G-protein-dependent signaling pathway by different experimental approaches, including the reconstitution of purified truncated receptors and G-proteins in phospholipid vesicles. Our data failed to provide evidence for IGF II-induced functional coupling and activation of G proteins through the M6P/IGF II receptor.


MATERIALS AND METHODS

Human recombinant IGF I and IGF II were generously supplied by Dr. W. Märki (Ciba Geigy, Basel) or were purchased from GroPep (Adelaide, SA). Phosphomannan from Hansenula hostii was a gift of Dr. M. E. Slodki (Northern Regional Research Center, Peoria, IL). Pentamannosyl 6-O-phosphate-substituted bovine serum albumin (PMP-BSA) was prepared and iodinated with the aid of IODO-GEN (24) to a specific activity of 65 µCi/µg. The following reagents were obtained commercially as indicated: TranS-label (ICN, Biomedical; 1100 Ci/mmol); [S]GTPS (1100-1400 Ci/mmol), [alpha-P]ATP, [P]phosphoric acid, and ^14C-labeled molecular mass standard proteins (DuPont-NEN); carrier-free NaI, [alpha-P]NAD (30 Ci/mmol), prestained high molecular standards, and the ECL detection reagents (Amersham Corp.); IODO-GEN and disuccinimidyl suberate (DSS) (Pierce), chloramine T, azolectin, mastoparan (Vespula leviesii), pertussis toxin, and CHAPS (Sigma); Pansorbin (10% staphylococcus aureus cell suspension) (Calbiochem). [-P]GTP was synthesized from [P]phosphoric acid as described(25) .

Oligonucleotides were synthesized on an Applied Biosystems model 381A solid phase synthesizer. The cDNA of human M6P/IGF II receptor and mouse L-cells deficient in M6P/IGF II receptor was kindly provided by Drs. William S. Sly (St. Louis University, St. Louis) and Stuart Kornfeld (Washington University, St. Louis).

Antibodies

The monoclonal antibody 2C2 directed against the human M6P/IGF II receptor (24) was iodinated with IODO-GEN (specific activity 8 µCi/µg). Antisera against the human liver M6P/IGF II receptor and the recombinant M6P/IGF II receptor tail were raised in goat and rabbit, respectively(26, 27) . Antiserum against the synthetic peptide corresponding to the last 15 C-terminal amino acids (peptide 15C) of the human M6P/IGF II receptor tail was obtained by immunizing a rabbit with the peptide coupled to keyhole limpet hemocyanine and was kindly provided by Dr. Annette Hille-Rehfeld (Institut für Biochemie II). Peptide antibodies AS 6 (anti-alpha), AS 8 (anti-alpha), AS 11 (anti-beta), AS 266 (anti-alpha), and AS 269 (anti-alpha) were described previously (28, 29, 30, 31) and kindly provided by Drs. Karsten Spicher and Klaus-Dieter Hinsch (Institut für Pharmakologie, Berlin).

Mutagenesis of M6P/IGF II Receptor

The human M6P/IGF II receptor cDNA was cleaved with KpnI, and a 4.6-kilobase fragment encoding the cytoplasmic tail, the transmembrane, and parts of the luminal domain (from base 3242 to 7851) was subcloned into M13mp18. Oligonucleotide-directed mutagenesis was carried out using single-stranded DNA (32) to introduce a stop codon at positions 2403 (CTG 224 TAG) and 2447 (CGT 224 TGA), which correspond to amino acids 75 and 119 of the cytoplasmic tail (StL 75 and StR 119, respectively). In the deletion mutant (D123-136) the codons for 14 amino acids (2451-2465, corresponding to amino acids 123-136 of the cytoplasmic tail) were deleted in frame. All mutations were verified by sequencing the final product. The wild type and mutant M6P/IGF II recetpor cDNA were subcloned in the expression vector pBHE.

Transfection of Cells

Mouse L-cells deficient for the M6P/IGF II receptor (L-) were maintained in a minimal essential medium (MEM) containing 5% fetal calf serum, 100 units/ml penicillin, and 100 µg/ml streptomycin at 37 °C in 5% CO(2). The cells were transfected with the pBHE plasmids and the pSV2neo plasmid (10:1) using the calcium phosphate technique. Selection was performed with 0.8 mg/ml G418. Stable colonies were isolated and screened for M6P/IGF II receptor expression by measuring the binding of iodinated 2C2 antibodies in saponin-permeabilized cells at 4 °C and by immunoprecipitation of receptors from cells metabolically labeled with TranS-label(24, 33) .

Purification of M6P/IGF II Receptors from Overexpressing Cells

Twenty 15-cm diameter dishes of confluent L-cells expressing human wild type or mutant M6P/IGF II receptors were used for each preparation. The cells were washed twice with ice-cold 10 mM phosphate-buffered saline, pH 7.4 (PBS) and scraped in 50 mM imidazole HCl, pH 7.4, containing 150 mM NaCl, 5 mM sodium beta-glycerophosphate, 5 mM EDTA, 3 mM iodoacetic acid, 0.04 trypsin inhibitory unit/ml aprotinin, 50 µM leupeptin, 1.25 mM phenylmethylsulfonyl fluoride, 50 mM NaF, and 0.05% bovine serum albumin (buffer A). All purification steps were carried out at 4 °C. The cells were spun at 1500 times g for 15 min, after which the pellets were resuspended in buffer A containing 0.1% saponin and 15 mM M6P. After a 5-min incubation on a rotating wheel the samples were spun at 1500 times g for 15 min followed by three additional extractions in the presence of saponin and M6P for 20 min each. The pellets were then washed 5 times for 15 min with buffer A containing 0.1% saponin and finally lysed in buffer A containing 1% Triton X-100 for 16 h. The samples were spun at 230,000 times g for 20 min in a Beckman TL-100 centrifuge, and the supernatants (approximately 60 mg of protein) were applied to a pentamannosyl phosphate-Sepharose 4B affinity column (3 ml, 0.2 ml/min) with three recycling steps. The column was washed with 50 ml of buffer A containing 1% Triton X-100 and with 10 ml each of buffer A containing 0.5, 0.1, or 0.05% Triton X-100. Thereafter the column was washed with 40 ml each of buffer A without aprotinin, leupeptin, phenylmethylsulfonyl fluoride, and BSA (buffer B); 50 mM Na-citrate buffer, pH 6.5, containing 75 mM NaCl, 100 mM P(i) and 0.05% Triton X-100; buffer B followed by 20 ml of buffer B, containing 100 mM glucose 6-phosphate (G6P). The receptors were eluted from the column with buffer B containing 20 mM M6P, concentrated, and dialyzed against 20 mM Hepes-NaOH, pH 7.0, containing 150 mM NaCl and 0.05% Triton X-100 in ultrafiltration thimbles (Schleicher & Schüll, exclusion size 75,000), which were pretreated with 1 M glycine solution, pH 7.4, to diminish the adhesion of receptors to the surface. The purity of M6P/IGF II receptors and the intactness of the cytoplasmic tails were assessed after SDS-PAGE by silver staining or Western blotting using different domain-specific receptor antibodies (see below). The amount of purified wild type or mutant receptor ranged from 20 to 30 µg depending on the preparation as determined by quantitative Western analysis using purified M6P/IGF II receptors from human placenta as standard.

Western Analysis

Aliquots of purified receptors or receptors reconstituted in phospholipid vesicles (see below) were subjected to SDS-PAGE (5% acrylamide) under reducing conditions. The proteins were transferred electrophoretically to nitrocellulose membrane (Bio Blot-NC, Costar, Cambridge) for 4 h at 4 °C at 900 mA (Transphor II, TE50X, Hoefer Scientific Instruments, San Francisco). Following transfer, the membranes were incubated for 1 h at 37 °C in 10 mM PBS containing 0.05% Triton X-100 and 5% (w/v) nonfat dry milk (buffer C). The membranes were then incubated for 18 h at 4 °C in buffer C and either human liver M6P/IGF II receptor-specific antiserum (1:1000 dilution), antiserum directed against the whole cytoplasmic receptor tail (1:100 dilution; (27) ), or an antiserum against peptide 15C (1:100 dilution). The membranes were rinsed five times in buffer C for 20 min and two times with 10 mM PBS containing 0.5% SDS for 15 min followed by extensive washing with distilled water and one wash with buffer C. The membranes were incubated for 2 h at room temperature with a goat anti-rabbit IgG coupled to peroxidase (1:50,000; Dianova, Hamburg). After 10 washes for 10 min each with buffer D (10 mM PBS containing 0.5% Triton X-100 and 0.2% SDS) membranes were rinsed in water and washed two times for 5 min with buffer E (10 mM Tris-HCl, pH 7.4, containing 150 mM NaCl and 0.05% Tween 20). Immunoreactive bands were visualized using the ECL detection system as recommended by the manufacturer.

Purification of G-proteins from Bovine Brain

Pertussis toxin-sensitive G-proteins were purified from bovine brains as described(34) . In brief, membranes were prepared and stored in 20 mM Tris-HCl, pH 8.0, containing 1 mM EDTA and 20 mM beta-mercaptoethanol (TEM) buffer at -70 °C. Thawed membranes were preextracted at 4 °C (as used for all procedures) for 1 h with TEM containing sodium cholate (0.1% w/v). The remaining material was extracted for 1 h with TEM supplemented with sodium cholate (0.9%). After centrifugation at 70,000 times g, the clear supernatant was mixed with ethylene glycol to a final concentration of 30% (v/v), and GDP (10 µM) was added. Purified mixtures of Galpha(o), Galpha(i)/Galpha(o), and Gbeta were obtained after sequential chromatography on a DEAE-Sepharose Fast Flow column (1.5 liters; Pharmacia Biotech Inc.), an AcA 34 gel filtration column (1.2 liters; Serva, Heidelberg, Germany), and a 0.65-liter heptylamine-Sepharose column(35) . Separation of alpha subunits from beta dimers was achieved as described(36) . G-proteins were identified by GTPS-binding, alpha and beta antibodies, and silver-stained polyacrylamide gels(34) . Individual Galpha subtypes of the G(i) subfamily, e.g. Galpha, Galpha, Galpha, Galpha, and Galpha, were resolved by chromatography on a Mono Q column (1 ml; Pharmacia) as reported(34) . Heterotrimeric forms of G, G, and G were isolated according to Codina et al.(37) . In addition, Galpha was also purified from human thrombocytes and Gbeta complexes from porcine brain. The G-proteins were detected after separation by SDS-PAGE (9% acrylamide) supplemented with 6 M urea in the separating gel followed by immunoblotting(31) . The membrane-bound antibodies were visualized by goat anti-rabbit alkaline phosphatase or goat anti-rabbit IgG coupled to peroxidase/ECL detection system.

Reconstitution of Purified M6P/IGF II Receptor and G-proteins in Phospholipid Vesicles

The purified wild type or mutant M6P/IGF II receptor protein (1 µg) and trimeric G or G (1 µg) were mixed with azolectin (1 mg/ml) and reconstituted by gel filtration (Sephadex G-50; 1.2 times 25 cm) as described(18) . To reconstitute the receptor with Galpha or Galpha and Gbeta the latter (2 µg) were mixed with the azolectin/cholate solution and kept on ice for 15 min followed by the addition of Galpha monomers (1 µg) for a further 15 min. After addition of receptor (1 µg) for 15 min and elution buffer (18) the mixture (600 µl in total) was applied onto a G-50 gel filtration column. Collected fractions were examined for I-IGF II binding and affinity cross-linking, [S]GTPS binding (specific activity 40 Ci/mmol), and GTPase activity as described below.

GTPS Binding Assay

G-proteins were quantitated by binding to [S]GTPS (500 nM) as described (34) . For receptor- or mastoparan-stimulated binding of [S]GTPS to G-proteins reconstituted vesicles (40 µl) were incubated with or without IGF II (1 µM) in buffer containing Hepes-NaOH, pH 7.4, 80 µM EDTA, 100 µM MgCl, and 100 nM [S]GTPS (specific activity, 200 Ci/mmol) in a total volume of 100 µl at 37 °C for the indicated times(19) . The reactions were terminated by adding 10 volumes of ice-cold buffer containing 20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 25 mM MgCl (TNM), and 20 µM GTP. The samples were filtered through a nitrocellulose filter (0.45 µm, Sartorius, Göttingen) washed three times with ice-cold TNM solution. After drying the filters were counted in an Ultima Gold scintillator (Canberra Packard).

Assay of GTPase Activity

The determination of GTPase activity was carried out as described(41) . Briefly, the reaction mixture contained 40 µl of solubilized membrane protein (5-10 µg) or reconstituted vesicles, 0.1 µM [-P]GTP (0.1 µCi/assay), 2 mM MgCl(2), 0.1 mM EDTA, 0.1 mM ATP, 5 mM phosphocreatine, 0.4 mg/ml creatine kinase, and the additions indicated in 50 mM of triethanolamine-HCl, pH 7.4, at 25 °C in a total volume of 100 µl. Low K(M) GTPase activity was calculated by subtracting high K(M) GTPase activity determined at 50 µM GTP from total GTPase activity. High K(M) GTPase activity was less than 10% of total radioactivity.

ADP-ribosylation of Membranes

Nontransfected L-cells and L-cells overexpressing wild type or mutant M6P/IGF II receptors were incubated for 1 h in serum-free medium before the addition of IGF II (30 nM) for 10 min at 37 °C. Membranes were prepared and resuspended in 50 mM Hepes-NaOH buffer, pH 7.4, containing 0.1 mM EDTA and 0.7% CHAPS at a final protein concentration of 20 mg/ml and immediately used for ADP-ribosylation (18) . The reaction was stopped by the addition of Laemmli buffer. After boiling for 5 min the samples were processed by SDS-PAGE (10% acrylamide) and analyzed by autoradiography. Alternatively, membranes were prepared from unstimulated cells and incubated with and without IGF II or GTPS at 37 °C for 15 min before initiation of the pertussis toxin (40 µg/ml) catalyzed ADP-ribosylation using [alpha-P] NAD as substrate(18) .

Other Methods

Human IGF I or IGF II was iodinated by the chloramine T method as described by Zapf et al.(38) . Nontransfected mouse L-cells, mouse L-cells overexpressing the wild type or mutant M6P/IGF II receptors, or aliquots of reconstituted receptors in phospholipid vesicles were incubated with I-IGF I or I-IGF II at 4 °C for 16 h and then cross-linked with DSS (0.06 mM) and analyzed by SDS-PAGE(39) . The protein content was measured by the Lowry procedure (40) . For ligand competition binding curves, L-cells expressing wild type or mutant M6P/IGF II receptors were incubated with I-PMP-BSA (1 pM) or I-IGF II (10 pM) in the absence or presence of various concentrations of nonradioactive ligands for 4 h at 4 °C(39, 42) .


RESULTS

Characterization of Expressed Wild Type and Mutant M6P/IGF II Receptors

Mutations were introduced in the cytoplasmic tail of the human M6P/IGF II receptor to generate C-terminal truncations of 45 (StR 119) and 89 (StL 75) amino acids as well as a third mutation lacking the proposed G-protein binding site (D 123-136). The cDNAs encoding the full-length receptor with either normal or mutant cytoplasmic tails were transfected together with the neomycin resistance gene in mouse L-cells, deficient for the M6P/IGF II receptor. Different G418-resistant colonies were isolated for each transfected cDNA, which were screened by antibody binding in permeabilized cells for expression of the M6P/IGF II receptors. Clones with similar expression levels (70-95% of the wild type receptor expression) were used in these experiments. Immunoprecipitation of M6P/IGF II receptors from metabolically labeled, transfected cells are shown in Fig. 1A. Truncation of the cytoplasmic tail resulted in increased electrophoretic mobility of the StR 119 and StL 75 receptor polypeptides. The distribution of M6P/IGF II receptors between cell surface and intracellular membranes was analyzed in a number of cell lines by I-labeled receptor antibody (2C2) binding in the presence or absence of 0.1% saponin, respectively. Nine percent of wild type M6P/IGF II receptors were present on the cell surface, compared with 12 and 14% of receptors truncated by one-fourth (StR 119) or having the deletion (D123-136), respectively. The percentage of StL 75 receptor mutants at the cell surface was increased to 22% of total cellular receptors, as shown previously by immunoelectron microscopy(43) . Immunofluorescence studies also revealed that the intracellular localization of the receptors was similar (not shown). When wild type receptor-expressing cells were incubated with I-PMP-BSA or I-IGF II at 4 °C in the presence or absence of increasing concentrations of unlabeled PMP-BSA and IGF II, half-maximal inhibition occurred at 18 ± 3 pM and 1.1 ± 0.4 nM, respectively. Nonspecific binding in the presence of 5 mM M6P or 0.1 µM IGF II was between 8 and 15% of cell-associated radioactivity. In cell lines expressing the mutant receptors, the binding properties varied in the same range.


Figure 1: Immunoprecipitation of M6P/IGF II receptors and affinity cross-linking of I-IGF II and I-IGF I to nontransfected and transfected mouse L-cells. A, L-cells expressing wild type or mutant M6P/IGF II receptors were metabolically labeled with [S]methionine. The receptors were immunoprecipitated by antibodies directed against the luminal domain and analyzed by SDS-PAGE and fluorography. B, cells were incubated with 3 ng I-IGF II or I-IGF I in the absence or presence of unlabeled IGF II or IGF I (0.5 µg), respectively. The receptor-ligand complexes were cross-linked with DSS and electrophoresed on a 7.5% polyacrylamide gel under reducing conditions. An autoradiogram of a representative experiment is shown. The positions of the M6P/IGF II, the IGF I receptor, and molecular mass markers (kDa) are indicated.



The specificity of I-IGF II binding to mutant cell surface receptors was investigated by affinity cross-linking. For this purpose, cells were incubated with I-labeled IGF II or IGF I, and then treated with DSS followed by SDS-PAGE under reducing conditions. Cross-linking with I-IGF II resulted in exclusive labeling of a 250-kDa band in wild type and D123-136 mutant M6P/IGF II receptor-expressing L-cells, whereas no 250-kDa cross-link product was observed in nontransfected L-cells (Fig. 1B). Under the conditions used, only faint binding of I-IGF II to the 135-kDa subunit of the IGF I receptor was detectable in nontransfected cell lines. The binding of I-IGF II to the receptor was inhibited by an excess of unlabeled IGF II. In L-cells expressing the truncated M6P/IGF II receptor mutants I-IGF II bound specifically to electrophoretically faster moving polypeptides than the wild type receptor polypeptides (not shown) corresponding to the electrophoretic pattern in Fig. 1A. After cross-linkage of I-IGF I bound to cell surface receptors of nontransfected and transfected mouse L-cells, in all cell lines one prominent labeled band of apparent 135 kDa was detectable representing the IGF I receptor subunit (Fig. 1B). No labeling of the M6P/IGF II receptor with I-IGF I was observed. The binding of I-IGF I to the 135-kDa protein was completely inhibited in the presence of an excess of unlabeled IGF I. These results indicate that the wild type and mutant M6P/IGF II receptors expressed in receptor-deficient cell lines are normally distributed and that the binding properties of mutant receptors for both classes of ligands were comparable with those of wild type receptors. Furthermore, IGF II bound specifically only to the M6P/IGF II and not to the IGF I receptor.

Effect of IGF II on Pertussis Toxin-catalyzed ADP-ribosylation of G(i)-proteins and GTPase Activity

To evaluate whether IGF II induces the activation of G-proteins we analyzed the ability of Bordetella pertussis toxin (Ptx) to modify G-proteins in cell membranes. Ptx has been reported to catalyze the ADP-ribosylation of some trimeric G-proteins (G(t), G(o), G(i))(44) . Ligand-induced subunit dissociation, therefore, should result in Ptx substrate activity. The following experiments parallel those reported by Okamoto et al.(45) who used EGF-primed, PDGF-competent, or ras-transformed BALB/c 3T3 cells to investigate IGF II-induced M6P/IGF II receptor G-protein coupling. The membranes were prepared from nontransfected, wild type, and D123-136 mutant M6P/IGF II receptor-expressing L-cells following incubation with IGF II. In mouse L-cell membranes, pertussis toxin catalyzes the [P]ADP-ribosylation of a 40-kDa protein (Fig. 2). After a 10-min treatment of the cell lines with 30 nM IGF II no significant changes in the pertussis toxin substrate activity were observed. In addition, preincubation of cells with IGF II or IGF I up to 100 nM for 5-30 min did not attenuate the extent of Ptx-catalyzed ADP-ribosylation of the 40-kDa protein (not shown). Furthermore, no alterations in the 40-kDa protein modification were detected after concomitant incubation of the different cell lines with IGF II and M6P or G6P (Fig. 2) or after incubation with the phosphorylated sugars alone (not shown). When cells were preincubated with Ptx (0.1 µg/ml) for 16 h the subsequent [P]ADP-ribose incorporation in the 40-kDa protein was inhibited by about 75% (Fig. 2).


Figure 2: Effect of IGF II on pertussis toxin substrate activity in nontransfected and transfected L-cells. Nontransfected, wild type, and D123-136 mutant M6P/IGF II receptor-expressing L-cells were incubated with 30 nM IGF II alone or in combination with 5 mM M6P or G6P for 10 min. The prepared membranes (100 µg of protein) were incubated with 40 µg/ml Ptx and 10 µM [P]NAD at 30 °C for 30 min. Proteins were electrophoresed by SDS-PAGE containing 10-15% acrylamide. As a control, membranes were prepared from cells preincubated for 16 h with Ptx (0.1 µg/ml). The molar mass of the Ptx substrate activity is indicated.



In a second approach, membranes prepared from different transfected and nontransfected L-cell lines were preincubated with 30 nM IGF II followed by Ptx-catalyzed ADP-ribosylation of the 40-kDa protein. This approach was carried out according to the experimental procedures of Nishimoto et al.(18) examining the effect of IGF II preincubation of BALB/c 3T3 membranes on the subsequent Ptx-catalyzed modification of the 40-kDa protein. Fig. 3indicates that IGF II did not cause a significant reduction in Ptx-substrate activity in any of the tested cell lines. Reduced ADP-ribosylation of the 40-kDa protein in membranes from L-cells expressing the truncated M6P/IGF II receptor StR 119 was observed in only one out of three experiments. In contrast, coincubation of membranes with GTPS, which activates G-proteins, reduced incorporation of [P]ADP-ribose into the 40-kDa proteins significantly by 60% (not shown). The pretreatment of membranes with Ptx and unlabeled NAD for 16 h inhibited the subsequent [P]ADP-ribose incorporation in the 40-kDa protein completely (Fig. 3). Whereas it was reported that in experiments carried out with intact BALB/c 3T3 cells (45) or with membranes prepared from these cells(18) , IGF II attenuates the Ptx-catalyzed ADP-ribosylation of the 40-kDa protein by 30-50 and 80%, respectively, in the present study IGF II failed completely to affect the ADP-ribose incorporation into the 40-kDa protein. Alternatively, we measured GTPase activity in membranes of nontransfected and wild type M6P/IGF II receptor-expressing L-cells preincubated with and without pre- or concomitant incubation of IGF II. None of the tested conditions revealed any significant stimulatory effect of IGF II on GTPase activity (not shown).


Figure 3: Effect of IGF II on pertussis toxin-catalyzed ADP-ribosylation in membranes of nontransfected and transfected L-cells. Membranes were prepared from nontransfected, wild type, and mutant M6P/IGF II receptor-expressing L-cells. The membranes were pretreated with IGF II (15 min) or Ptx (16 h), centrifuged, and resuspended (20 mg/ml) before ADP-ribosylation in the presence of 40 µg/ml Ptx and 10 µM [P]NAD for 30 min at 30 °C. The proteins were separated by SDS-PAGE (10-15% acrylamide) and visualized by autoradiography.



Additionally, in EGF ``primed'', PDGF ``competent'' L-cells (45) expressing the wild type M6P/IGF II receptor, IGF II did not attenuate the Ptx-catalyzed ADP-ribosylation of the 40-kDa protein (not shown). The 40-kDa pertussis toxin substrate in mouse L-cells was recognized by the anti-Galpha antibody AS 269 and comigrated with purified ADP-ribosylated Galpha but not with pertussis toxin-modified Galpha or Galpha (data not shown). All of these results indicate that in M6P/IGF II receptor-expressing L-cells, IGF II failed to activate G-proteins.

Purification and Characterization of Wild Type and Truncated M6P/IGF II Receptors Expressed in L-cells

Mouse L-cells expressing the human wild type or truncated M6P/IGF II receptors were solubilized with Triton X-100 and passed over a phosphomannan-Sepharose affinity column. The fractions eluted with M6P contained only one protein as shown by silver staining (Fig. 4A) with an apparent molecular mass of 250 kDa. The identity of the protein as the M6P/IGF II receptor was confirmed by Western immunostaining using an antibody directed against the luminal receptor domain (Fig. 4B). To determine whether the purified wild type receptor contained an intact C terminus, its reactivity with an antibody directed against the last 15 amino acids of the human M6P/IGF II receptor (peptide 15C) was tested. It should be noted that when the purification was carried out in the presence of divalent cations (1 mM) despite protease inhibitors, the cytoplasmic tail of the receptor was partially or completely degraded. The addition of EDTA and various protease inhibitors yielded a wild type M6P/IGF II receptor with a complete cytoplasmic tail (Fig. 4C). The purified, truncated M6P/IGF II receptors showed the characteristic gel shift, compared with wild type receptor, when analyzed by silver or immunostaining using an antibody directed against the luminal domain of the M6P/IGF II receptor; whereas the anti-15C antibody did not react with the truncated receptor. The purified truncated receptors still contained parts of the cytoplasmic tail confirmed by their reactivity with a polyclonal antibody directed against the entire cytoplasmic receptor tail expressed in Escherichia coli ((27) ; data not shown).


Figure 4: Purification of wild type and truncated M6P/IGF II receptors. A, a Triton X-100 extract of mouse L-cells expressing the human wild type or truncated M6P/IGF II receptor was passed over a phosphomannan-Sepharose column. An aliquot of the M6P eluate (1 µg of protein) was separated on SDS-PAGE followed by silver staining. B and C, a portion of the M6P-eluted receptor fraction was immunostained after Western blotting by means of antibodies directed against the luminal domain (B) or against C-terminal peptide 15C (C).



Reconstitution of M6P/IGF II Receptor and G-proteins in Phospholipid Vesicles

Human wild type M6P/IGF II receptor purified from overexpressing mouse L-cells by M6P-affinity chromatography and trimeric G purified from bovine brain (Fig. 5) were reconstituted in phospholipid vesicles. The gel chromatographic procedure used for reconstitution was identical to that described by Nishimoto and colleagues(18) . Protein-containing vesicles eluted in the void volume and collected fractions were analyzed. Usually, out of four fractions, two showed the highest GTPS binding activity (Fig. 6A). This was in accordance with the basal and mastoparan-stimulated GTPase activity of the fractions tested (Fig. 6B). Incubation of reconstituted vesicles with mastoparan (100 µM) stimulated the GTPase activity about 6-10-fold. Mastoparan also stimulated significantly the binding of GTPS to G by about 2-fold (see Fig. 7C), confirming its ability to activate the reconstituted G, analogous to hormone receptor-mediated G-protein activation(20) . Mean reconstitution efficiency was 39 ± 16% (n = 16). Reconstitution of functional, active M6P/IGF II receptors was shown by I-IGF II affinity cross-linking followed by immunoprecipitation with polypeptide 15C antibody directed against the C terminus of the receptors (Fig. 6C). The binding of I-IGF II to the receptor was inhibited by an excess of unlabeled IGF II. The results showed an identical elution profile of receptors as compared with reconstituted G-proteins. The co-insertion of M6P/IGF II receptors with G-proteins did not affect the total GTPS binding or GTPase activity as compared with vesicles containing G or G alone (data not shown). These data provide compelling evidence that the vesicles contain functional G proteins and ligand binding-competent M6P/IGF II receptors with complete cytoplasmic domains. Similar results were obtained after reconstitution of wild type or StL 75 and StR 119 mutant M6P/IGF II receptors with trimeric G or G, or Galpha or Galpha, together with purified Gbeta. The two fractions containing the highest amount of G-proteins and receptors were pooled and used immediately to study the functional coupling of the M6P/IGF II receptor to G-proteins.


Figure 5: Identification and pertussis toxin-mediated ADP-ribosylation of G and G. Aliquots of fractions containing purified G- (A) and G-proteins (B) were precipitated with acetone in the presence of BSA (10 µg) and loaded on gels (6 M urea, 9% acrylamide). After blotting, nitrocellulose filters were incubated with antibodies AS 6 (anti-alpha serum, diluted 1:300), AS 248 (anti-alpha antibodies, affinity-purified), AS 266 (anti-alpha antibodies, affinity-purified), AS 269 (anti-alpha serum, diluted 1:150). For ADP-ribosylation, G-protein alpha-subunits (Galpha, 600 ng; Galpha, 340 ng) were treated with pertussis toxin in the presence of unlabeled and P-labeled NAD (lanes 5 and 6) and brain beta subunits (680 ng) as described under ``Materials and Methods.'' Proteins were subsequently resolved on gels (6 M urea, 9% acrylamide). After blotting, nitrocellulose-filters were exposed to x-ray films. Corresponding alpha-subunits were detected by incubating nitrocellulose filters with antibody AS 6 (anti-alpha serum diluted 1:300, A, lanes 1-3) or AS 266 (anti-alpha antibodies, affinity-purified; B, lanes 1-3). The ECL system was used for detection of filter-bound antibodies. A, lane 1, heterotrimeric G(i)/G(o)-pool; lane 2, heterotrimeric G; lanes 3-6, Galpha. B, lane 1, heterotrimeric G(i)/G(o)-pool; lane 2, heterotrimeric G; lanes 3-6, Galpha. Molecular masses (in kDa) of marker proteins are indicated. It should be noted that pertussis toxin-mediated ribosylation of Galpha (A, lanes 5 and 6) was incomplete, resulting in two immunoreactive bands (lane 5) of unmodified (lower band) and modified (upper band) protein.




Figure 6: Characterization of G protein and M6P/IGF II receptors reconstituted into phospholipid vesicles. A, [S]GTPS (500 nM, 100,000 cpm/tube) binding to G in different phospholipid vesicle containing fractions eluted from the Sephadex G-50 column. B, GTPase activity. Reconstituted vesicles were incubated with (black bars) or without (open bars) 100 µM mastoparan for 15 min at 25 °C prior to determination of GTP hydrolysis as described under ``Materials and Methods.'' C,I-IGF II affinity cross-linkage followed by receptor immunoprecipitation. Reconstituted vesicles were incubated with I-IGF II in the presence and absence of unlabeled IGF II (0.5 µg) for 16 h at 4 °C. After cross-linkage with DSS and immunoprecipitation using antibodies directed against the 15 C-terminal amino acids of the cytoplasmic M6P/IGF II receptor domain, the immunocomplexes were analyzed by SDS-PAGE (5% acrylamide) and autoradiography. The position of the 200-kDa molecular mass standard is indicated. Panels A-C show the data of one representative experiment out of three to five for each of the tested trimeric G-proteins. The data for panels A and C were obtained from the same preparation.




Figure 7: Effect of IGF II and mastoparan on GTPS binding to G or G in reconstituted vesicles. Wild type (A, B, and C), StL 75 (D and E), or StR 119 mutant (F) M6P/IGF II receptors reconstituted with trimeric G (A and D) or G (B, C, E, and F) were incubated for the indicated period with [S]GTPS in the absence (circle) or presence (bullet) of 1 µM IGF II or 100 µM mastoparan (). Each point represents the mean of duplicates. The GTPS binding to G-proteins of one representative experiment out of two to four is shown. GTPS binding values were corrected by the unspecific binding in the presence of 100 µM unlabeled GTPS.



Effect of IGF II on GTPS Binding of G-proteins in Reconstituted Vesicles

Wild type M6P/IGF II receptors reconstituted with the trimeric forms of either G or G were incubated with 1 µM IGF II for up to 60 min. IGF II increased the rate of GTPS binding to receptor/G or receptor/G vesicles to a maximum of 1.2- or 1.3-fold after a 5-20-min incubation (Fig. 7, A and B). The incubation of wild type receptor/G vesicles with 100 µM mastoparan stimulated significantly the GTPS binding rate 2.2-fold after 5 min and 1.6-fold after 10 min (Fig. 7C). Similarly, in reconstituted vesicles containing the StL 75 receptor mutant, which lacks the putative G-protein binding site and G trimers, the GTPS binding was also increased at most 1.2-1.3-fold in response to IGF II (Fig. 7E). In StR 119/G or StL 75/G vesicles, IGF II did not affect the GTPS binding rate as compared with controls (Fig. 7, D and F). Maximal GTPS binding to the different G-protein-containing vesicles was reached after 30-60 min of incubation and was not altered by IGF II. Similar results were obtained after reconstitution of M6P/IGF II receptors with mixtures of purified G(i)/G(o) proteins (not shown). We further reconstituted wild type M6P/IGF II receptors with purified monomer Galpha or Galpha from bovine brain and human thrombocytes and beta dimers from bovine or porcine brain (alpha:beta ratio of 1:2). In both cases, the rate of GTPS binding was not affected by IGF II over a time course of 60 min and was comparable with that seen in the trimeric G-protein experiments (Fig. 8). Similar results were obtained in experiments carried out with two to four different wild type or mutant receptor preparations, one preparation of G, and two preparations of G as well as with two batches of recombinant human IGF II from two different sources. In wild type receptor/G vesicles prepared by the Extracti-Gel procedure(46) , 1 µM IGF II also failed to stimulate the GTPS binding (data not shown).


Figure 8: Effects of IGF II on GTPS binding to Galpha monomers and beta dimers reconstituted vesicles. Wild type M6P/IGF II receptors were reconstituted with Galpha or Galpha monomers and beta dimers in an alpha:beta ratio of 1:2. The vesicles were incubated for the indicated period with [S]GTPS in the absence (circle) or presence (bullet) of 1 µM IGF II. Each point is the mean of duplicates.



Effect of IGF II on GTPase Activity of G in Wild Type and Mutant M6P/IGF II Receptor Reconstituted Vesicles

As a second means to study activation of G by IGF II, we analyzed receptor-mediated stimulation of G GTPase activity. In reconstituted G vesicles containing either the wild type or the StL 75 M6P/IGF II receptor mutant, IGF II (1 µM) failed to stimulate the GTPase activity of G. Addition of mastoparan (100 µM) for 15 min increased the GTPase activity 7-11-fold of both reconstituted vesicle populations (Fig. 9).


Figure 9: Effects of IGF II and mastoparan on GTPase activity of G in reconstituted vesicles. Wild type (n = 3) or StL 75 mutant (n = 2) M6P/IGF II receptors reconstituted with trimeric G were incubated for 15 min at 25 °C with [P]GTP in the absence or presence of IGF II (1 µM) or mastoparan (100 µM). All values represent means of two to three determinations carried out in triplicate. The data are expressed in relation to untreated controls that account for 236 ± 43 fmol of P(i) released per fraction and min.



Taken together, in reconstituted vesicles containing different G-proteins and wild type or truncated M6P/IGF II receptors devoid of the putative G-protein binding site, IGF II increased the GTPS binding only slightly (1.2-1.3-fold) independent of the subtype of G-protein used (G or G) and the presence of the proposed G-protein-coupling sequence in the cytoplasmic receptor tail. In addition, in reconstituted vesicles containing wild type M6P/IGF II receptors and purified Galpha(i) and Galpha(o) monomers and beta-dimers, IGF II did not alter the GTPS binding. Finally, in wild type or mutant M6P/IGF II receptor/G vesicles, IGF II did not induce a stimulation of GTPase activity. These results fail to support the proposal that IGF II binding to M6P/IGF II receptors induces the functional coupling and activation of G proteins via amino acid residues 123-136 of the cytoplasmic receptor tail.


DISCUSSION

Whereas the IGF I receptor is capable of mediating the transmembrane signaling of both IGF I and IGF II ligands by activation of its intrinsic tyrosine kinase, it was previously proposed that the binding of IGF II to the M6P/IGF II receptor activates G proteins. The M6P/IGF II receptor does not share structural properties with conventional seven-transmembrane G-coupled receptors but instead contains a sequence of 14 amino acids in its cytoplasmic domain which was proposed to activate G proteins directly(18, 21) . The experiments presented here examine interactions between wild type and mutant M6P/IGF II receptors with G-proteins in response to IGF II. In this work we followed the two main experimental approaches that were initially used (18, 19, 45) to implicate the direct coupling of M6P/IGF II receptors with G proteins: i) pertussis toxin-catalyzed ADP-ribosylation of a 40-kDa protein in cell membranes and ii) GTPS binding to and GTPase activity of purfied G-proteins in reconstituted phospholipid vesicles containing purified M6P/IGF II receptors in response to IGF II.

In the first approach, we investigated the effect of IGF II to reduce the ability of pertussis toxin to modify G(i) proteins in intact mouse L-cells expressing the human wild type M6P/IGF II receptor, nontransfected L-cells deficient for the M6P/IGF II receptor, or L-cells expressing a mutant receptor lacking the proposed G-protein activator sequence motif. Pertussis toxin modifies only trimeric GDP-bound G(i) proteins by ADP-ribosylation, whereas the dissociated GTP-bound alpha subunits formed after ligand-induced G-protein activation do not function as toxin substrates(44) . The 40-kDa pertussis toxin substrate of L-cells is Galpha, as demonstrated by the pertussis toxin-catalyzed ADP-ribosylation of L-cell membranes in comparison with the ADP-ribosylation of purified G, G, and G proteins as well as anti-Galpha antibody reactivity. This is in accordance with other reports that assume that G is a major pertussis toxin-sensitive G-protein in non-neuronal cells(43, 47, 48, 49) . In all of the tested cells, neither IGF II nor IGF I was able to reduce pertussis toxin-catalyzed ADP-ribosylation of Galpha over a wide range of concentrations. In addition, no attenuation of pertussis toxin substrate activity was observed in broken L-cell membranes containing the human wild type or D 123-136 M6P/IGF II receptor mutant. Neither in our present study nor in studies using BALB/c 3T3 cells were positive controls tested, i.e. the ability of ligands of known G(i) protein-coupled receptors (such as the m2 muscarinic receptor) to attenuate the Ptx-catalyzed ADP-ribosylation. First, it is difficult to test such controls in L-cells and second, it is questionable whether ligand-induced activation of G(i) proteins can diminish the amount of Ptx substrate activity. Thus it is rather unlikely that in the presence of GTP a ligand-induced transient dissociation of G-proteins might affect the extent of the Ptx-catalyzed ADP-ribosylation, which rapidly takes place whether or not the G- protein is undergoing dynamic cycling.

We used additional criteria to confirm the inability of IGF II to induce the activation and dissociation of a pertussis toxin substrate: (i) inability of IGF II to stimulate GTPase activity in nontransfected or M6P/IGF II-overexpressing L-cells, (ii) inability of IGF II to attenuate the pertussis toxin-catalyzed ADP-ribosylation in PDGF/EGF-treated (primed competent) L-cells expressing the wild type M6P/IGF II receptor, and (iii) receptor-independent GTPS-induced attenuation of pertussis toxin substrate activity. All of these data indicate that the binding of IGF II to the M6P/IGF II receptor expressed in mouse L-cells does not initiate transmembrane signaling via activation of G. This is in contrast to reports that incubation of BALB/c 3T3 cell membranes with either IGF II or IGF I (5-10 nM) led to a reduction in the pertussis toxin substrate activity by 70-80% within 10 min(18) . Additionally, in intact BALB/c 3T3 cells, low concentrations of IGF II and IGF I (1 nM) reduced the pertussis toxin substrate activity of G by 50% but with slightly differing time courses(45) . However, both IGFs did produce changes in pertussis toxin substrate activity of G but only in primed competent BALB/c 3T3 cells, i.e. those pretreated for 3 h with PDGF followed by a 20-min EGF treatment or in v-ras p21-transformed BALB/c 3T3 cells.

The authors postulate that (i) in quiescent cells the M6P/IGF II receptors are uncoupled from G resulting in nonresponsiveness to IGF II and (ii) competence growth factors or v-ras p21 activation restores functional coupling between the M6P/IGF II receptor and G, which enables both IGFs to stimulate calcium influx in these cells. If the mouse L-cells have some mechanism for coupling the M6P/IGF II receptor with G, it must be different from that observed in BALB/c 3T3 cells since coupling could not be obtained by treatment with PDGF/EGF. However, it remains unclear why intact BALB/c 3T3 cells should require pretreatment with competence growth factors to detect attenuation of pertussis toxin substrate activity in response to IGF II, whereas IGF II would be capable of activating G proteins directly in membranes prepared from quiescent BALB/c 3T3 cells via the M6P/IGF II receptor. Finally, a different cellular composition of beta dimers in M6P/IGF II receptor-deficient mouse L-cells used in our mutational analysis could not be excluded, which could affect the response to IGF II (see below).

To circumvent these problems, we turned to a second approach, which utilize a controlled reconstituted system containing purified components. In these experiments potential M6P/IGF II receptor-G-protein interaction in phospholipid vesicles was followed by measuring GTPS binding and GTPase activity in response to IGF II. The human M6P/IGF II receptors used in these experiments were purified from overexpressing mouse L-cells lacking the M6P/IGF II receptor. During purification under optimized conditions, no proteolytic cleavage of the cytoplasmic receptor tail occurred, as monitored by binding of antibodies directed against the carboxyl-terminal 15 amino acids. We demonstrated that the wild type M6P/IGF II receptors and the G-proteins were functionally active in reconstituted vesicles. However, in vesicles containing the wild type M6P/IGF II receptor, neither G, G, nor purified G(i)/G(o) mixtures yielded a significant stimulation of GTPS binding or GTPase activity in response to IGF II. Furthermore, similar findings were observed in experiments in which truncated receptor proteins lacking the proposed G-protein activator sequence of the cytoplasmic receptor domain were reconstitued with G or G proteins or mixtures of all G(i)/G(o) proteins. IGF II failed to increase significantly the binding of GTPS or GTPase activity. In contrast, 100 µM mastoparan yielded an approximately 2-fold increase in GTPS binding and 9-fold increase in GTPase activity in vesicles reconstituted with wild type or StL 75 receptor and G trimers. These results differ from those of Nishimoto and colleagues(18, 19) , who reported that IGF II induced a functional coupling and activation of Galpha with purified M6P/IGF II receptors reconstituted in phospholipid vesicles (2-fold increase in the GTPS binding rate and GTPase activity), which were completely inhibited by M6P and the M6P-containing glucuronidase.

Although we have no obvious explanation for the discrepancies between their findings and ours, we must consider possible differences in the G-proteins or M6P/IGF II receptors employed. Nishimoto's group used G-proteins purified from a variety of sources (bovine spleen, lung, brain, or porcine brain)(18, 19, 50) , although a similar purification procedure was used in both studies. Furthermore, differences in beta composition of the G-protein preparation used in the two laboratories, which might facilitate or modulate stability, localization, and activation of subunits or might directly interact with receptors (51, 52) can also be excluded because the purified beta complexes used here represent a mixture of beta dimers most likely identical with beta complexes Nishimoto employed. Therefore it seems more likely that the M6P/IGF II receptor preparations of the two laboratories were different. Whereas proteolytic cleavage of the cytoplasmic domain of M6P/IGF II receptors was prevented during our purification procedure, Nishimoto did not characterize the employed receptor preparations in great detail. This might be particularly important with regard to proposed suppressor sequences within the cytoplasmic receptor domain that could attenuate G-protein activation in the absence of IGF II (53) . The removal of such a suppressor element, localized C-terminal to the proposed G-protein-activating sequence by proteolytic cleavage during preparation, could facilitate the IGF II-induced G-protein activation. The loss of parts of the cytoplasmic domain of M6P/IGF II receptors during purification from bovine liver was previously reported (54) . Alternatively, reversible posttranslational modifications of the cytoplasmic M6P/IGF II receptor domain, such as phosphorylation/dephosphorylation, might modulate the functional responsiveness to IGF II. This could explain the requirement of PDGF/EGF pretreatment or ras p21 transformation of quiescent BALB/c 3T3 cells to restore coupling of receptors to G(45, 17) . It has been shown that following treatment of serum-deprived BALB/c 3T3 cells with different growth factors, the cellular cAMP levels increased (55) resulting in protein kinase A activation. There are several reports of typical G-protein-coupled receptors phosphorylated in an agonist-dependent manner by receptor kinases resulting in desensitization of these receptors(50, 56, 57) . Thus, the loss of phosphate groups from distinct sites during the purification procedure could contribute to a preformed IGF II-responsive M6P/IGF II receptor conformation.

The physiological relevance of the proposed IGF II-induced activation of G is still a matter of speculation. Whereas some reports observed the generation of intracellular signals in response to IGF II, such as the activation of phospholipase C, calcium influx, and DNA synthesis(15, 17) , which are also stimulated by IGF I at low concentrations(45, 58, 59) , other studies failed to demonstrate changes in intracellular levels of inositol phosphates, diacylglycerols, cAMP, or calcium after incubation with IGF II (60, 61, 62, 63) . Furthermore, analysis of mutant mouse embryos indicated that only the IGF I receptor and an additional as yet unknown receptor that is not the M6P/IGF II receptor mediate the mitogenic signaling of IGF II(7, 13) .

At the moment we cannot exclude the possibility that the M6P/IGF II receptor coupling to G-proteins and the stimulation of signal transduction pathways in response to IGF II are cell type-specific and require additional cellular activation processes. However, the localization of only 5-15% of total M6P/IGF II receptors at the cell surface and their rapid replacement within 2.8 min (24) as compared with typical G-protein-coupled signaling receptors argues against a role of M6P/IGF II receptors in signaling. Alternatively, trimeric G-proteins might be involved in regulation of cellular trafficking of M6P/IGF II receptors (for review see (64) ).

Activation of G(i) proteins has been shown to inhibit vesicle budding from the TGN(65) . It is not known whether the M6P/IGF II receptor functions as an activator of G-proteins in the TGN and thereby affects budding and vesicular transport to endosomes. There are no data showing that precursor or mature forms of IGF II bind in the TGN to M6P/IGF II receptors, and the binding of M6P-containing newly synthesized lysosomal enzymes does not activate G-proteins(19) . It remains to be determined whether phosphorylation of the M6P/IGF II receptor cytoplasmic domain by a TGN-associated receptor kinase (66) or the receptor association with cytosolic/membrane-bound proteins (27) may contribute to G-protein activation.

In conclusion, the present study fails to support a model in which the M6P/IGF II receptor functionally couples to G-proteins in cell membranes or in phospholipid vesicles.


FOOTNOTES

*
This study was supported by the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 236/B 11 and Sonderforschungsbereich 366) and the Fonds der Chemischen Industrie. 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: Georg-AugustUniversität Göttingen, Gosslerstr. 12d, D-37073 Göttingen, Federal Republic of Germany. Tel.: 49-551-395902; Fax: 49-551-395979.

(^1)
The abbreviations used are: M6P, mannose 6-phosphate; IGF II, insulin-like growth factor II; BSA, bovine serum albumin; PMP-BSA, pentamannose 6-phosphate coupled to BSA; PAGE, polyacrylamide gel electrophoresis; G-protein, heterotrimeric regulatory guanine nucleotide-binding protein; GTPS, guanosine 5`-0-(3-thiotriphosphate); Ptx, pertussis toxin; PBS, phosphate-buffered saline; G6P, glucose 6-phosphate; EGF, epidermal growth factor; PDGF, platelet-derived growth factor; TGN, trans-Golgi network.


ACKNOWLEDGEMENTS

We are grateful to William S. Sly (St. Louis University, St. Louis, MO) and Stuart Kornfeld (Washington University, St. Louis, MO) for generously providing the cDNA of the human M6P/IGF II receptor and mouse L-cells, respectively. We also thank Ikno Nishimoto (Harvard Medical School) for providing his laboratory protocol for performing the reconstitution assay. Moreover, we thank Kurt von Figura for encouraging the work and Suzan Pfeffer, Elliot M. Ross, and Stuart Kornfeld for critical reading of the manuscript and thoughtful comments. Finally, we thank Martin Lohse (Max-Planck-Institute, Munich) for help with the Extracti-Gel reconstitution procedure.


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