Coordinate Regulation of the α2-Macroglobulin Signaling Receptor and the Low Density Lipoprotein Receptor-related Protein/α2-Macroglobulin Receptor by Insulin*

We have studied insulin-dependent regulation of macrophage α2-macroglobulin signaling receptors (α2MSR) and low density lipoprotein receptor-related protein/α2M receptors (LRP/α2MR) employing cell binding of 125I-α2M*, inhibition of binding by receptor-associated protein (RAP) or Ni2+, LRP/α2MR mRNA levels, and generation of second messengers. Insulin treatment increased the number of α2M* high (α2MSR) and low (LRP/α2MR) affinity binding sites from 1,600 and 67,000 to 2,900 and 115,200 sites per cell, respectively. Neither RAP nor Ni2+ blocked the binding of125I-α2M* to α2MSR on insulin- or buffer-treated cells, but they both blocked binding to LRP/α2MR. Insulin significantly increased LRP/α2MR mRNA levels in a dose- and time-dependent manner. Insulin-augmented125I-α2M* binding to macrophages was severely reduced by wortmannin, LY294002, PD98059, SB203580, or rapamycin. The increase in α2MSR receptor synthesis was reflected by augmented generation of IP3 and increased [Ca2+] i levels upon receptor ligation. Incubation of macrophages with wortmannin, LY294002, PD98059, SB203580, rapamycin, or antibodies against insulin receptors before insulin treatment and α2M* stimulation significantly reduced the insulin-augmented increase in IP3 and [Ca2+] i levels. Pretreatment of cells with actinomycin D or cycloheximide blocked the synthesis of new α2MSR. In conclusion, we show here that insulin coordinately regulates macrophage α2MSR and LRP/α2MR, utilizing both the PI 3-kinase and Ras signaling pathways to induce new synthesis of these receptors.

Human ␣ 2 -macroglobulin (␣ 2 M) 1 is a homotetrameric proteinase inhibitor present at high concentration in blood and tissue fluids (1,2). ␣ 2 M reacts with endoproteinases of every mechanistic class in a reaction that induces a major conformational change in the inhibitor. This change exposes receptor recognition sites in the carboxyl terminus of each of its four subunits, leading to rapid clearance from the circulation and in vitro binding by cells expressing receptors for receptor-recognized forms of ␣ 2 M (␣ 2 M*) (2,3). Small nucleophiles such as methylamine directly attack internal ␤-cysteinyl-␥-glutamyl thiolesters present in each subunit causing a similar conformational change in the inhibitor with comparable exposure of receptor recognition sites (1)(2)(3). Low density lipoprotein receptor-related protein/␣ 2 M receptor (LRP/␣ 2 MR) is a high molecular weight cell surface receptor expressed by many cell types including macrophages, fibroblasts, hepatocytes, adipocytes, and dermal dendritic cells (2)(3)(4)(5). LRP/␣ 2 MR is a scavenger receptor (K d ϳ5 nM) that binds multiple structurally and functionally diverse ligands besides ␣ 2 M*, including Pseudomonas exotoxin A, lipoproteinase lipase, apolipoprotein E-enriched lipoproteins, urokinase, and tissue-type plasminogen activator alone or in combination with plasminogen activator inhibitor-1, tissue factor pathway inhibitor, and lactoferrin (2,5). Generally, these ligands do not compete with each other for binding to LRP/␣ 2 MR, presumably because they bind to independent receptor domains; however, receptor-associated protein (RAP, M r ϳ39,000) blocks the binding of all known ligands to this receptor (4,5). In addition to LRP/␣ 2 MR, ␣ 2 M* binds to a recently discovered ␣ 2 M signaling receptor (␣ 2 MSR) (K d ϳ50 pM) present on a more restricted range of cells than LRP/␣ 2 MR (5)(6)(7)(8)(9)(10)(11)(12)(13)(14). Binding of ␣ 2 M* to LRP/␣ 2 MR is followed by uptake and degradation in lysosomes but not activation of a signaling cascade (6,7,12). By contrast, binding of ␣ 2 M* or its receptor binding fragment to ␣ 2 MSR triggers typical signaling cascades, which regulate cell proliferation (6 -15). RAP and Ni 2ϩ prevent ␣ 2 M* binding to LRP/␣ 2 MR, but do not inhibit the ability of ␣ 2 M* to bind to ␣ 2 MSR, and they do not affect signal transduction (16). Based on a variety of evidence, we have proposed that ␣ 2 MSR behaves like various growth factor receptors and that it is involved in cellular growth regulation (6 -14).
A relationship between insulin and 125 I-␣ 2 M* receptors was first reported from this laboratory by Ney et al. (36), who showed that pretreatment of adipocytes and fibroblasts with insulin enhanced the binding of 125 I-␣ 2 M* to cell surface receptors and decreased receptor-mediated degradation of ␣ 2 M by fibroblasts. These observations were later confirmed by others (37)(38)(39)(40). Certain growth factors, for example insulin and nerve growth factors as well as cytokinins like interleukin-I␤ and tumor necrosis factor-␣, also up-regulate the synthesis of LRP/ ␣ 2 MR and low density lipoprotein receptors in neurons (41) and HepG2 cells (42). In the latter case, activation of ERK1 and -2 was required for cytokinin-induced increased expression of low density lipoprotein receptors (42). In the present study, we have studied the effect of insulin on expression of macrophage LRP/␣ 2 MR and ␣ 2 MSR by the following parameters: 1) binding of 125 I-␣ 2 M* to high (␣ 2 MSR) and low (LRP/␣ 2 MR) affinity binding sites on the cells and generation of second messengers, namely IP 3 and Ca 2ϩ ; 2) RAP and Ni 2ϩ sensitivity of binding of 125 I-␣ 2 M* to ␣ 2 MSR and LRP/␣ 2 MR on buffer-or insulintreated cells; 3) sensitivity of 125 I-␣ 2 M* binding and second messenger generation to PI 3-kinase inhibitors, wortmannin and LY294002, ERK1/2 inhibitor PD98059, p38 MAPK inhibitor SB203580, and p70 s6k inhibitor rapamycin; and 4) LRP/␣ 2 MR receptor mRNA levels. Based upon these criteria, we report here that insulin appears to regulate the expression of macrophage LRP/␣ 2 MR and ␣ 2 MSR in a coordinate manner.

EXPERIMENTAL PROCEDURES
Materials-Brewer's thioglycollate broth was purchased from Difco. Culture media were purchased from Life Technologies, Inc. Bovine serum albumin, pertussis toxin, staurosporin, insulin, cycloheximide, actinomycin D, genestein, HEPES, and rapamycin were purchased from Sigma. Fura-2/AM was obtained from Molecular Probes, Inc. (Eugene, OR). Myo[2-3 H]inositol (specific activity 10 -20 Ci/mmol) was purchased from American Radiolabeled Chemicals (St. Louis, MO). Insulin receptor antibody was purchased from Immunotech (Westbrook, ME). A plasmid containing the RAP cDNA was a kind gift from Dr. Joachim Herz (University of Texas, Southwestern, Dallas, TX). It was used to produce RAP as described previously (11). [␣-32 P]dCTP and 125 I were purchased from NEN Life Science Products. Iodobeads® were purchased from Pierce. Wortmannin, LY294002, PD98059, and SB203580 were obtained from Biomol (Plymouth Meeting, PA). Human ␣ 2 M was purified, converted to ␣ 2 M* with methylamine, and radiolabeled with 125 I as described previously (6,12). All other reagents used were of the highest grade available.
Insulin and Macrophage Binding of 125 I-␣ 2 M*-Pathogen-free C57BI/6 mice (6 weeks old) were obtained from Charles River (Raleigh, NC). Binding of 125 I-␣ 2 M* to thioglycollate-elicited macrophages was studied as detailed earlier (12,13,16). 125 I-␣ 2 M* was added to cells in wells at varying concentrations and incubated for 2 h at 4°C. Free ligand was separated from bound by aspirating the medium and monolayers carefully washed five times with ice-cold RPMI 1640 medium containing penicillin, streptomycin, and 2% bovine serum albumin (buffer A). The cells were lysed with 1 M NaOH at 40°C, and bound activity was determined in a ␥-counter. The specific binding of 125 I-␣ 2 M* was calculated by subtracting nonspecific binding determined in the presence of 5 mM EDTA (12,13,16,43). To study the effect of insulin on 125 I-␣ 2 M* binding, insulin (10 nM) was added to monolayers in buffer A, and monolayers were incubated for specific periods of time at 37°C, transferred to ice, and washed five times with ice-cold buffer A. 125 I-␣ 2 M* was added to wells at the specified concentrations, monolayers were incubated for 2 h at 4°C, and binding was determined as described above. The effects of PI 3-kinase inhibitor wortmannin (30 nM/30 min/ 37°C) or LY294002 (20 M/15 min/37°C) on the binding of 125 I-␣ 2 M* to insulin-or buffer-treated macrophages was determined by addition to the monolayers prior to incubation with insulin (10 nM/1 h/37°C). Cells were then washed, and binding of 125 I-␣ 2 M* was studied as outlined above. In experiments where the effects of PD98059 (50 M/90 min/ 37°C), SB203580 (15 M/30 min/37°C), or rapamycin (100 nM/5 min/ 37°C) were studied on insulin-augmented cellular binding of 125 I-␣ 2 M*, these were added to monolayers prior to adding insulin (10 nM/1 h/37°C), and binding of 125 I-␣ 2 M* was determined as outlined above. Protein in cell lysates was determined by the Bradford method (44). K d values were calculated using the Sysstat Program as described previously (13).
Determination LRP/␣ 2 MR mRNA Levels-The effect of insulin on macrophage LRP/␣ 2 MR mRNA was determined as described earlier (13). A partial human cDNA fragment ranging from base pair 188 to 6,179 inserted into plasmid pGEM 4 was used for hybridization with mRNA isolated from insulin-treated and -untreated macrophages. This plasmid was a kind gift of Dr. Joachim Herz.
Measurement of IP 3 in Insulin-treated Cells-The formation of IP 3 in [ 3 H]myoinositol-labeled macrophages under various experimental conditions was quantified essentially according to methods published earlier (6 -10). In experiments where insulin effects were studied on ␣ 2 M*induced changes in IP 3 levels, the cells were treated with insulin at the specified concentrations or time periods, and insulin was washed out with HHBSS before stimulating with ␣ 2 M*. In studies where the effects of cycloheximide, actinomycin D, genestein, or staurosporin were studied on changes in IP 3 in cells pretreated with insulin followed by stimulation with ␣ 2 M*, these agents were added at the specified concentration and incubated for the specified time period at 37°C prior to the addition of insulin or buffer. The effects of wortmannin, LY294002, PD98059, SB203580, or rapamycin also were examined on insulinaugmented increased IP 3 formation of ␣ 2 M*-stimulated cells. Macrophages labeled as above were incubated with the respective inhibitors, before the addition of insulin (10 nM/1 h/37°C). The cells were incubated for 1 h, washed with HHBSS buffer containing 10 mM Li ϩ , 1 mM Ca 2ϩ , and 1 mM Mg 2ϩ , followed by stimulation with ␣ 2 M* and quantification of IP 3 as described above.
Insulin Receptor Antibody and Insulin-induced Changes in IP 3 -These studies were performed as outlined above except that washed [ 3 H]myoinositol-labeled macrophages (3 ϫ 10 6 cells/4.5 cm 2 ) in a volume of RPMI 1640 medium containing 0.25% bovine serum albumin, leupeptin (20 g/ml), and phenylmethylsulfonyl fluoride (1 mM) were incubated with antibody against insulin receptors (10 g/ml) for 30 min at 25°C. At the end of incubation, insulin (10 nM) or buffer was added to monolayers, and the incubation continued for an additional 60 min at 37°C. The monolayers were washed five times with ice-cold HHBSS buffer containing Li ϩ , Ca 2ϩ , and Mg 2ϩ , and cells were incubated in this buffer for 5 min at 37°C prior to stimulation with ␣ 2 M* followed by quantification of inositol phosphates (6 -10).

Measurement of [Ca 2ϩ ] i in Insulin-treated Cells-Changes in [Ca 2ϩ
] i levels in Fura-2/AM-loaded single cells were quantified using digital imaging microscopy as described earlier (6 -10). Briefly, cells were incubated with the specified concentrations of insulin for the desired time periods and loaded with Fura-2/AM in the last 30 min of incubation. Monolayers were washed with HHBSS and stimulated with ␣ 2 M*, and changes in [Ca 2ϩ ] i were measured as described (6 -10). In experiments where the effects of insulin were studied on ␣ 2 M*-induced changes in [Ca 2ϩ ] i levels, the cells were treated with insulin at specified concentrations or time periods, and insulin was washed out with HHBSS before stimulating with ␣ 2 M*. The effects of cycloheximide, actinomycin D, genestein, or staurosporin on changes in [Ca 2ϩ ] i were studied in cells pretreated with insulin followed by stimulation with ␣ 2 M*. These agents were added at specified concentrations and incubated for the specified period of time at 37°C prior to the addition of insulin or buffer.

RESULTS
Insulin Enhances the Binding of 125 I-␣ 2 M* to both ␣ 2 MSR and LRP/␣ 2 MR-Insulin treatment increased the overall binding of 125 I-␣ 2 M* to macrophages by about 1.5-2-fold compared with buffer-treated cells (Fig. 1A). By Scatchard analysis, the K d for ligand binding to the high affinity binding site (␣ 2 MSR) was 50 pM, and the number of binding sites per cell was 1,600 (Fig. 1B). The K d for ligand binding to the low affinity binding sites (LRP/␣ 2 MR) was 25 nM, and the number of binding sites per cell was 67,000 (Fig. 1B). Incubation of macrophages with insulin prior to binding of ligand had little effect on the K d of binding to either site, but it increased the number of both the high affinity (2,800) and the low affinity (115,200) sites, respectively. RAP (10-fold excess) did not block binding to the high affinity sites in buffer-or insulin-treated macrophages, but it reduced binding to the low affinity site for the ligand by about 50% in both buffer and insulin-treated cells (Fig. 1B). Like RAP, Ni 2ϩ , which inhibits the binding of ␣ 2 M* to LRP/␣ 2 MR but not to ␣ 2 MSR (16), also inhibited the binding of 125 I-␣ 2 M* by about 60% in both buffer-and insulin-treated cells (Fig. 1C). The maximal binding of 125 I-␣ 2 M* occurred when cells were exposed to 10 nM insulin, while at lower or higher concentrations, insulin treatment markedly reduced ligand binding (Fig. 2).
Insulin Increases mRNA Levels of LRP/␣ 2 MR-The maximum increase in LRP/␣ 2 MR mRNA levels occurred when cells were treated with 10 nM insulin, but above or below this insulin concentration, a marked decrease in mRNA was observed (Fig.  3A). LRP/␣ 2 MR expression was maximal at 1 h of incubation with 10 nM insulin (Fig. 3B). The presence of actinomycin D before and during insulin treatment nearly abolished the insulin-induced increase in receptor mRNA, demonstrating that the increased mRNA level observed in insulin-treated cells was due to insulin-induced new RNA synthesis (Fig. 3B). (45) and LY294002 (46), both inhibitors of PI 3-kinase, reduced insulinaugmented increased cellular binding of 125 I-␣ 2 M* without significantly altering basal ligand binding (Fig. 4A). PI 3-kinase activity is required for activation of p70 s6k , which plays a potential role in regulating protein synthesis by phosphorylation of ribosomal S6 kinase (47). Treatment of cells with rapamycin, an inhibitor of p70 s6k (48), before insulin treatment reduced the insulin-augmented increased binding of 125 I-␣ 2 M* without significantly altering the basal binding of 125 I-␣ 2 M* to macrophages (Fig. 4B). Activation of the Ras pathway is the second route of signal transduction that has been implicated in the induction of gene expression by insulin (49). Components of the pathway that includes ERK1, ERK2, and p38 MAPK are mediators of phosphorylation of intracellular substrates such as protein kinases and transcription factors as well as regulators of growth factor-induced cell growth and differentiation (50,51). Treatment of cells with PD98059, a specific inhibitor of ERK1 and ERK2 (52), or SB203580, a specific inhibitor of p38 MAPK (53), before insulin treatment, like the PI 3-kinase and p70 s6k inhibitors significantly decreased the insulin-augmented increased binding of 125 I-␣ 2 M* to macrophages without significantly affecting basal binding of the ligand (Fig. 4B). These results support the hypothesis that insulin increased the binding of 125 I-␣ 2 M* to macrophages by activating both the PI 3-kinase and Ras signaling pathways to achieve this synthesis.

PI 3-Kinase and MAPK Inhibitors Reduce Insulin-induced Increased Binding of 125 I-␣ 2 M* to Cells-Wortmannin
Insulin-induced Increase in IP 3 (6 -15). Incubation of macrophages with insulin prior to stimulation with ␣ 2 M* increased IP 3 levels in a biphasic manner (Fig. 5). In insulin-treated cells, a 2-4-fold increase in IP 3 formation was observed in the initial 60 s of agonist stimulation, followed by another sustained increase of up to 5 min compared with buffer-treated cells (Fig. 5A). The generation of IP 3 after ␣ 2 M* stimulation of insulin-pretreated macrophages was proportional to the concentration of insulin up to 10 nM, but incubation of macrophages with higher concentrations of insulin caused a gradual decrease in IP 3 formation (Fig. 5B). The maximal stimulation of IP 3 synthesis occurred after 1 h of insulin treatment, and a decrease in IP 3 formation occurred after longer treatment. Insulin by itself showed no effect on the generation of IP 3 in macrophages under the experimental conditions. Preincubation of macrophages with actinomycin D or cycloheximide before insulin treatment showed little effect on the ␣ 2 M*-induced early increase in IP 3 levels, but this treatment abolished the insulin-induced sustained increase in IP 3 levels (Fig. 6A). The addition of actinomycin D after incubation of macrophages with insulin followed by stimulation with ␣ 2 M* had no effect on insulin-induced increase in IP 3 . These results suggest that the potentiated generation of IP 3 in insulintreated cells arises as a consequence of the increased number of newly synthesized ␣ 2 MSR. Treatment of macrophages with genestein (a tyrosine kinase inhibitor) or staurosporin (a protein kinase C inhibitor) prior to incubation with insulin followed by stimulation with ␣ 2 M* abolished the augmented increase in IP 3 production (Fig. 6B). Thus, the involvement of protein kinase C and tyrosine kinases in insulin-induced upregulation of ␣ 2 MSR and second messenger events following ␣ 2 MSR activation is evident. 3 Generation-To demonstrate that the effects of insulin on the binding of ␣ 2 M* and generation of IP 3 occur consequent to its binding to insulin receptors on macrophages, we next studied the effect of incubating macrophages with insulin receptor antibody before adding insulin (Fig. 6B). As expected, treatment of macrophages with antibody against membrane insulin receptors abolished the insulin-induced aug-mented increase in IP 3 generation seen upon ␣ 2 M* stimulation, but it showed very little effect on IP 3 generation in macrophages treated with buffer prior to stimulation with ␣ 2 M* (Fig.  6B). These results show that the increased level of IP 3 observed on ␣ 2 M* stimulation of insulin-treated macrophages results from first binding of insulin to its receptor followed by sequential activation of downstream signal transduction events, resulting in up-regulation of ␣ 2 MSR.

Inhibition of Insulin-induced Increase in IP 3 Formation by PI 3-Kinase and MAPK Pathways
Inhibitors-We next evaluated the involvement of the PI 3-kinase and MAPK pathways in the insulin-induced up-regulation of ␣ 2 MSR by quantifying the generation of IP 3 in cells treated with PI 3-kinase pathway inhibitors wortmannin and LY294002, and MAPK pathway inhibitors PD98059 and SB203580, and the p70 s6k inhibitor rapamycin before insulin treatment and ␣ 2 M* stimulation (Fig.  7). As expected, inhibitor treatment significantly reduced only the insulin-augmented increase in IP 3 generation but not basal levels observed upon stimulation with ␣ 2 M* (Fig. 7). These results suggest that the potentiated increase in IP 3 synthesis observed in insulin-treated macrophages occurs due to the availability of increased numbers of receptors and that insulin utilizes both PI 3-kinase and MAPK pathways downstream of insulin stimulation to induce this new synthesis of ␣ 2 MSR.
Effect of Insulin on [Ca 2ϩ ] i Levels in ␣ 2 M*-stimulated Macrophages-Many extracellular stimuli cause an increase in cytosolic [Ca 2ϩ ] i by stimulating formation of IP 3 , which then binds to the intracellular IP 3 receptor, an ion channel. This causes its opening and release of Ca 2ϩ from intracellular membranebound compartments. This is followed by the entry of Ca 2ϩ from the extracellular medium by capacitative Ca 2ϩ entry mechanisms (54). Treatment of macrophages with insulin prior to stimulation with ␣ 2 M* increased [Ca 2ϩ ] i levels by 2-3-fold compared with buffer-treated macrophages (Fig. 8A). In a typical experiment, [Ca 2ϩ ] i levels in unstimulated cells, ␣ 2 M*stimulated cells, and insulin-treated and ␣ 2 M*-stimulated cells were 144.51 Ϯ 4.58, 356.78 Ϯ 13.79, and 836.18 Ϯ 15.80 nM, respectively. The increase in [Ca 2ϩ ] i levels was proportional to insulin concentration, being maximal at 10 nM and showing a slight decrease thereafter (Fig. 8B). As would be predicted, the effect of insulin concentration on [Ca 2ϩ ] i levels upon ␣ 2 M* stimulation is very similar to the augmented increase in IP 3 levels that was observed (Fig. 5). Increase in [Ca 2ϩ ] i on ␣ 2 M* stimulation of insulin-treated macrophages was maximal at 1 h of insulin treatment, and longer incubation resulted in a slight decrease in [Ca 2ϩ ] i levels. Incubation of macrophages with actinomycin D, cycloheximide, genestein, or staurosporin in separate experiments before treatment with insulin and stimulation with ␣ 2 M* suppressed the insulin-augmented increase in [Ca 2ϩ ] i levels but only slightly affected ␣ 2 M*-induced increases in [Ca 2ϩ ] i levels in buffer-treated macrophages (Fig. 8C). DISCUSSION The primary observations of this study are that insulin treatment of peritoneal macrophages prior to stimulation with ␣ 2 M* are as follows. 1) Insulin treatment increases the number of both high (␣ 2 MSR) and low (LRP/␣ 2 MR) affinity binding sites by about 2-fold. 2) Treatment augments the increase in two second messengers, namely IP 3  both LRP/␣ 2 MR and ␣ 2 MSR. These results contrast to previous suggestions that the insulin-augmented increase in the binding of 125 I-␣ 2 M* to LRP/␣ 2 MR occurred because of redistribution of receptors (36 -39), decreased endocytosis (36 -39, 55), or increased half-life of receptor-ligand complex (36).
Two mechanisms by which insulin exerts its metabolic and mitogenic effects after binding to receptors are activation of PI 3-kinase and its downstream signaling pathway and activation of the small GTP binding protein p21 ras and its downstream protein kinases (17)(18)(19)(20)(21)(22). Inhibition of PI 3-kinase activity by wortmannin and LY294002 attenuates the stimulation of glucose transport and mitogenesis by insulin (27). This lipid kinase is linked to the regulatory cascade that controls p70 s6k and activation by insulin, which can be selectively blocked by rapamycin (26,56). The p21 ras pathway has been implicated in the induction of gene expression by insulin (17-21, 26, 57). The expression of dominant inhibitory mutants of p21 ras inhibited insulin-stimulated gene expression (26). Selective inhibition of farnesyl protein transferase prevented the attachment of Ras proteins to plasma membranes and adversely affected their activation by growth factors (58). In macrophages, ligation of ␣ 2 MSR results in activation of the p21 ras (35) and PI 3-kinase (34) signaling pathways, and we have proposed that these mechanisms are involved in ␣ 2 M*-induced mitogenesis (13,14).
In most cells, insulin signaling extends to the nucleus with changes in transcription of a variety of specific genes (20,21,32,59). Insulin receptors have been identified in nuclei (60,61) causing stimulation of nuclear tyrosine kinase activity, which could be important in insulin action at the nuclear level (60 -62). Insulin regulates the transcription and turnover of mRNAs that encode proteins involved both in growth and metabolic processes (17)(18)(19)(20)(21)(22), stimulates phosphorylation of a number of transcription factors, and DNA-binding proteins (20 -22, 32, 59), and increases the levels of several specific mRNAs including that of the p33 gene, c-fos, c-jun, early growth response genes, lipoprotein lipase (20,21,32,59). The present work demonstrates that a coordinate up-regulation of LRP/␣ 2 MR and ␣ 2 MR by insulin also occurs. The potentiated, biphasic increase in IP 3 generation in cells treated with insulin first and subsequently stimulated with ␣ 2 M* is largely attributed to the availability of increased number of newly synthesized ␣ 2 MSR molecules available for occupancy.
When insulin-treated cells are exposed to a ␣ 2 M*, it binds to two pools of ␣ 2 MSR, one a constitutive/basal pool, unaffected by actinomycin D, and the second a newly synthesized pool, which is greatly reduced by actinomycin D, cycloheximide, genestein, staurosporin, PI 3-kinase inhibitors, MAPK pathway inhibitors, and antibodies against insulin receptors. The insulinexpanded and constitutive/basal receptor pools, upon ␣ 2 M* binding, trigger PIP 2 hydrolysis, generating IP 3 that, upon binding to IP 3 receptors, causes higher levels of [Ca 2ϩ ] i . These events trigger the onset of several intracellular signaling cascades that act in synergy with those activated by insulin consequent to its binding to receptors and thus enhance the cellular responses. Based upon the similarities in second messenger generation elicited upon ligation of ␣ 2 MSR with those elicited upon ligation of growth factor receptors with their cognate ligands, we have hypothesized that ␣ 2 MSR is a growth factorlike receptor whose ligation with receptor-recognized forms of ␣ 2 M generates a series of signaling events that play a role in growth and that this receptor may be involved in tissue repair. Thus, in the presence of a physiological concentration of insulin, the synergy or "cross-talk" between insulin and ␣ 2 MSR may be physiologically important.