Terminal amino acid sequences and proteolytic cleavage sites of mouse mammary tumor virus env gene products

The mature envelope glycoproteins of mouse mammary tumor virus (gp52 and gp36) were isolated by reversed-phase high-pressure liquid chromatography. The N-terminal amino acid sequence of gp36 was determined for 28 residues. The C-terminal amino acid sequences of gp52 and gp36 were determined by carboxypeptidase digestion. The N-terminal amino acid sequence of gp52 has been reported previously (L. O. Arthur et al., J. Virol. 41:414-422, 1982). These data were aligned with the predicted amino acid sequence of the env gene product obtained by translation of the DNA sequence (S. M. S. Redmond and C. Dickson, Eur. Mol. Biol. Org. J. 2:125-131, 1983). The amino acid sequences of the mature viral proteins were in agreement with the predicted amino acid sequence of the env gene product over the regions of alignment. This alignment showed the sites of proteolytic cleavages of the env gene product leading to the mature viral envelope glycoproteins. The N-terminal amino acid sequence of gp52 starts at residue 99 of the predicted structure indicating proteolytic cleavage of a signal peptide. A dipeptide (Lys-Arg) is excised between the C-terminus of gp52 and the N-terminus of gp36. The C-terminal amino acid sequence of gp36 is identical to the sequence predicted by the codons immediately preceding the termination codon for the env gene product. The data show that there is no proteolytic processing at the C-terminal of the murine mammary tumor virus env gene product and that the env gene coding region extends into the long terminal repeat.     

Control of amino acid transport in the mammary gland of the pregnant mouse

The regulation of the uptake of the amino acid analog α-aminoisobutyric acid was studied in diced mammary glands from pregnant mice. Stimulation of uptake by insulin was not prevented by inhibitors of protein synthesis; protein synthesis inhibitors decreased uptake by 20%; this response occurred more promptly in insulintreated tissues. Elimination of extracellular amino acids led to a substantial increase in transport which was not abolished by inhibitors of protein synthesis. These results indicate that insulin does not increase amino acid transport in this system by altering synthesis and degradation of transport protein. They are consistent with a model in which the activity of the existing amino acid transport protein is subject to negative feedback regulation from the intracellular amino acid pool.     

Protein kinase C activators selectively inhibit insulin-stimulated system A transport activity in skeletal muscle at a post-receptor level.

We have investigated the role of phorbol esters on different biological effects induced by insulin in muscle, such as activation of system A transport activity, glucose utilization and insulin receptor function. System A transport activity was measured by monitoring the uptake of the system A-specific analogue alpha-(methyl)aminoisobutyric acid (MeAIB), by intact rat extensor digitorum longus muscle. The addition of 12-O-tetradecanoylphorbol 13-acetate (TPA, 0.5 microM) for 60 or 180 min did not modify basal MeAIB uptake by muscle, suggesting that insulin signalling required to stimulate MeAIB transport does not involve protein kinase C activation. However, TPA added 30 min before insulin (100 nM) markedly inhibited insulin-stimulated MeAIB uptake. The addition of polymyxin B (0.1 mM) or H-7 (1 mM), protein kinase C inhibitors, alone or in combination with TPA leads to impairment of insulin-stimulated MeAIB uptake. This paradoxical pattern is incompatible with a unique action of Polymyxin B or H-7 on protein kinase C activity. Therefore these agents are not suitable tools with which to investigate whether a certain insulin effect is mediated by protein kinase C. TPA did not cause a generalized inhibition of insulin action. Thus both TPA and insulin increased 3-O-methylglucose uptake by muscle, and their effects were not additive. Furthermore, TPA did not modify insulin-stimulated lactate production by muscle. In keeping with this selective modification of insulin action, treatment of muscles with TPA did not modify insulin receptor binding or kinase activities. In conclusion, phorbol esters do not mimic insulin action on system A transport activity; however, they markedly inhibit insulin-stimulated amino acid transport, with no modification of insulin receptor function in rat skeletal muscle. It is suggested that protein kinase C activation causes a selective post-receptor modification on the biochemical pathway by which insulin activates system A amino acid transport in muscle.     

Phorbol ester differentially regulates oxytocin receptor binding activity in hypothalamic cultured neurons and astrocytes

Hypothalamic cultured neurons and astrocytes were used to investigate the cellular mechanisms underlying the oxytocin receptor-mediated downregulation through a possible involvement of protein kinase C (PKC). For this purpose, the effects of PKC activators, inhibitor and of OT on OT receptor binding activity were compared in both cultures. In neurons, phorbol-myristate-acetate (PMA), a potent PKC activator, increased the binding of an OT receptor antagonist whereas in astrocytes, a decrease was observed. Pre-treatment of the cells with bisindolylmaleimide (10(-4) M), a PKC inhibitor, prevented the PMA-induced up- and downregulation. In contrast, receptor downregulation resulting from treatment of both cells with OT (10(-9) M) was not affected by the PKC inhibitor. On the other hand, when PMA (10(-7) M) was tested along with OT (10(-9) M), a subsequent decrease in ligand binding was observed in astrocytes. In neurons, PMA attenuated the OT-induced downregulation. Structural analysis of neuron and astrocyte OT receptor mRNA by RT-PCR, subcloning and sequencing, demonstrated identical sequence to rat uterine receptor. In conclusion, these data suggest that activation of PKC has opposite effect on OT receptor binding activity in neurons and astrocytes but they do not support the involvement of PKC in the OT-induced downregulation.     

Detection and characterization of mouse mammary tumor virus cell surface antigens: estimation of antigen abundance by protein A assay.

Antisera against the following mouse mammary tumor virus (MMTV) structural proteins were used to detect MMTV cell surface antigens: (i) the 27,000-dalton nucleoid protein, p27; (ii) the 36,000-dalton envelope glycoprotein, gp36; and (iii) the 52,000-dalton exterior envelope glycoprotein, gp52. We report here the development of an adherent-cell isotopic staphylococcal protein A (SPA) test (ISPAT) for MMTV structural proteins which allows for the detection of an MMTV membrane-associated antigen as well as an estimate of its relative abundance on the cell surface. This test demonstrated that the gp52 was the predominant MMTV cell surface antigen detected on both C3H and GR mouse mammary tumor cells. In a comparative study with anti-gp52 and anti-gp36 sera, SPA-specific binding with anti-gp36 serum was found to be only 5 to 6% of that obtained for the external virion glycoprotein, gp52. Both direct and indirect ISPAT indicated the presence of a low but detectable number of gp36 determinants on GR-MMTV cells; however, these gp36 determinants, unlike gp52 determinants, appeared to be exposed by the fixation procedure used. Only 0.9 to 1.1% of the gp52-specific binding was detected when anti-gp36 serum was allowed to react with viable cells. The binding of [125I]SPA achieved with anti-p27 serum was even less than that detected with gp36-directed reagents, indicating that p27 is not a cell surface antigen. The use of fluoresceinated SPA further demonstrated that p27 and gp36 reactivity was only associated with a small number of cells in each of the mammary cultures tested. When N-[4-(5-nitro-2-furyl)-2-thiazoly]-formamide-induced C3H bladder tumor cells were subjected to a gp52-directed ISPAT, the failure to detect gp52-specific binding demonstrated the specificity of this assay for MMTV gp52-expressing cells. In addition to detecting and characterizing MMTV cell surface antigens, the newly developed adherent cell assay could measure changes in the abundance of cell surface gp52. When dexamethasone-treated and untreated GR cells were compared, measurements of gp52-specific SPA binding indicated that dexamethasone stimulation leads to a 12.2-fold increase in the amount of cell surface gp52 detected.     

Effects of phorbol esters on insulin receptor function and insulin action in hepatocytes: evidence for heterogeneity

We investigated the effect of phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator on insulin receptors and insulin action in freshly isolated and primary cultures of rat hepatocytes. PMA (1 x 10^–7 M) did not alter insulin receptor numbers or affinity either acutely or chronically but within 60 minute inactivated insulin stimulated tyrosine kinase of the insulin receptor. PKC activation inhibitied insulin (1 x 10^–7M) stimulation of glycogen and lipid synthesis with a decrease or no change in basal glycogenesis and lipogenesis respectively. However, PKC activation did not alter insulin stimulated or basal amino acid transport even though PCK activation inhibited insulin stimulation of the insulin. receptor tyrosine kinase. Thus, within one tissue, PKC activation has differential effect on insulin action depending on which pathway is examined. Furthermore, insulin stimulation of the insulin receptor tyrosine kinase may not be a necessary step for all insulin signaling pathways.     

Processing and amino acid sequence analysis of the mouse mammary tumor virus env gene product.

The envelope proteins of mouse mammary tumor virus (MMTV) are synthesized from a subgenomic 24S mRNA as a 75,000-dalton glycosylated precursor polyprotein which is eventually processed to the mature glycoproteins gp52 and gp36. In vivo synthesis of this env precursor in the presence of the core glycosylation inhibitor tunicamycin yielded a precursor of approximately 61,000 daltons (P61env). However, a 67,000-dalton protein (P67env) was obtained from cell-free translation with the MMTV 24S mRNA as the template. To determine whether the portion of the protein cleaved from P67env to give P61env was removed from the NH2-terminal end of P67env and as such would represent a leader sequence, the NH2-terminal amino acid sequence of the terminal peptide gp52 was determined. Glutamic acid, and not methionine, was found to be the amino-terminal residue of gp52, indicating that the cleaved portion was derived from the NH2-terminal end of P67env. The NH2-terminal amino acid sequences of gp52's from endogenous and exogenous C3H MMTVs were determined though 46 residues and found to be identical. However, amino acid composition and type-specific gp52 radioimmunoassays from MMTVs grown in heterologous cells indicated primary structure differences between gp52's of the two viruses. The nucleic acid sequence of cloned MMTV DNA fragments (J. Majors and H. E. Varmus, personal communication) in conjunction with the NH2-terminal sequence of gp52 allowed localization of the env gene in the MMTV genome. Nucleotides coding for the NH2 terminus of gp52 begin approximately 0.8 kilobase to the 3' side of the single EcoRI cleavage site. Localization of the env gene at that point agrees with the proposed gene order -gag-pol-env- and also allows sufficient coding potential for the glycoprotein precursor without extending into the long terminal repeat.     

Hormonal regulation of protein kinase C in the mouse mammary gland

The hormonal regulation of protein kinase C (PKC) induction over 3 to 14 days was investigated in the mouse mammary gland in vitro and in vivo. In intact mice, estradiol (1 microgram/mouse injected daily for 2 weeks) stimulated PKC activity 70%, while progesterone (1 mg/mouse injected daily) inhibited it by 30%. Prolactin, whose levels were elevated for 2 weeks by two pituitary isografts, had no effect. When mammary gland explants were cultured in insulin and cortisol, the further addition of estradiol (1 ng/ml), progesterone (1 microgram/ml), or prolactin (1 microgram/ml) did not alter PKC activity after 3 days. These data suggest the following conclusions: although previous studies have implicated prolactin in the transient, calcium-phospholipid activation of PKC, it does not appear to elevate total levels of this kinase over prolonged periods. In contrast, the sex steroids do appear to affect long-term levels of this kinase; furthermore, this latter effect may be indirect.     

Characterization and regulation of the gene expression of amino acid transport system A (SNAT2) in rat mammary gland

Amino acid transport via system A plays an important role during lactation, promoting the uptake of small neutral amino acids, mainly alanine and glutamine. However, the regulation of gene expression of system A [sodium-coupled neutral amino acid transporter (SNAT)2] in mammary gland has not been studied. The aim of the present work was to understand the possible mechanisms of regulation of SNAT2 in the rat mammary gland. Incubation of gland explants in amino acid-free medium induced the expression of SNAT2, and this response was repressed by the presence of small neutral amino acids or by actinomycin D but not by large neutral or cationic amino acids. The half-life of SNAT2 mRNA was 67 min, indicating a rapid turnover. In addition, SNAT2 expression in the mammary gland was induced by forskolin and PMA, inducers of PKA and PKC signaling pathways, respectively. Inhibitors of PKA and PKC pathways partially prevented the upregulation of SNAT2 mRNA during adaptive regulation. Interestingly, SNAT2 mRNA was induced during pregnancy and to a lesser extent at peak lactation. beta-Estradiol stimulated the expression of SNAT2 in mammary gland explants; this stimulation was prevented by the estrogen receptor inhibitor ICI-182780. Our findings clearly demonstrated that the SNAT2 gene is regulated by multiple pathways, indicating that the expression of this amino acid transport system is tightly controlled due to its importance for the mammary gland during pregnancy and lactation to prepare the gland for the transport of amino acids during lactation.     

Insulin receptor substrate-2 phosphorylation is necessary for protein kinase C zeta activation by insulin in L6hIR cells

We have investigated glycogen synthase (GS) activation in L6hIR cells expressing a peptide corresponding to the kinase regulatory loop binding domain of insulin receptor substrate-2 (IRS-2) (KRLB). In several clones of these cells (B2, F4), insulin-dependent binding of the KRLB to insulin receptors was accompanied by a block of IRS-2, but not IRS-1, phosphorylation, and insulin receptor binding. GS activation by insulin was also inhibited by >70% in these cells (p < 0.001). The impairment of GS activation was paralleled by a similarly sized inhibition of glycogen synthase kinase 3 alpha (GSK3 alpha) and GSK3 beta inactivation by insulin with no change in protein phosphatase 1 activity. PDK1 (a phosphatidylinositol trisphosphate-dependent kinase) and Akt/protein kinase B (PKB) activation by insulin showed no difference in B2, F4, and in control L6hIR cells. At variance, insulin did not activate PKC zeta in B2 and F4 cells. In L6hIR, inhibition of PKC zeta activity by either a PKC zeta antisense or a dominant negative mutant also reduced by 75% insulin inactivation of GSK3 alpha and -beta (p < 0.001) and insulin stimulation of GS (p < 0.002), similar to Akt/PKB inhibition. In L6hIR, insulin induced protein kinase C zeta (PKC zeta) co-precipitation with GSK3 alpha and beta. PKC zeta also phosphorylated GSK3 alpha and -beta. Alone, these events did not significantly affect GSK3 alpha and -beta activities. Inhibition of PKC zeta activity, however, reduced Akt/PKB phosphorylation of the key serine sites on GSK3 alpha and -beta by >80% (p < 0.001) and prevented full GSK3 inactivation by insulin. Thus, IRS-2, not IRS-1, signals insulin activation of GS in the L6hIR skeletal muscle cells. In these cells, insulin inhibition of GSK3 alpha and -beta requires dual phosphorylation by both Akt/PKB and PKC zeta.     

Effects of trypsin on binding of insulin and concanavalin A and on glucose and proline transport in the R3230AC mammary adenocarcinoma

Effects of trypsin treatment on insulin and concanavalin A binding to, and glucose and proline transport in, dissociated R3230AC mammary adenocarcinoma cells were examined. Reduction of binding of ¹²⁵I-labelled insulin was dependent on the amount of trypsin used, the temperature and the time of the incubation period. Under conditions that reduced insulin binding by greater than 75%, transport of glucose and proline was reduced by less than 15%. Scatchard analysis of insulin binding after trypsin treatment yielded slopes similar to those from cells not exposed to trypsin, assuming either two classes of receptors or an average affinity, $\text{K}\text{?}{}_{\mathrm{<mtext>e</mtext>}}$. Dissociation of bound insulin from untreated or trypsin-treated cells was enhanced by addition of excess unlabelled ligand. Insulin added in vitro, which decreased glucose transport in untreated cells, produced a decrease in glucose transport in cells treated with trypsin for 5 min (insulin binding was decreased 35%), but not in cells treated for 45 min (insulin binding was decreased 90%). Binding of the plant lectin concanavalin A was also reduced by trypsin treatment, but to a lesser extent and with a different time-course than for insulin. Scatchard analysis of the binding of concanavalin A in untreated and trypsin-treated cells yielded comparable values for K_d. The insulinomimetic actions of concanavalin A on glucose transport were abolished after brief exposure to trypsin. Pre-treatment of cells with concanavalin A reduced insulin binding and partially protected insulin receptors from trypsin digestion, but the inability to remove all of the concanavalin A precluded its use as a method to protect insulin receptors. Thus, in this rat mammary tumor, the number, but not the affinity or functional activity, of insulin receptors can be reduced by trypsin treatment without significant effects on glucose or A system amino acid transport.     

Protein kinase C inhibitors alter neurotensin receptor binding and function in prostate cancer PC3 cells

Prostate cancer PC3 cells expressed constitutive protein kinase C (PKC) activity that under basal conditions suppressed neurotensin (NT) receptor function. The endogenous PKC activity, assessed using a cell-based PKC substrate phosphorylation assay, was diminished by PKC inhibitors and enhanced by phorbol myristic acid (PMA). Accordingly, PKC inhibitors (staurosporine, Go-6976, Go-6983, Ro-318220, BIS-1, chelerythrine, rottlerin, quercetin) enhanced NT receptor binding and NT-induced inositol phosphate (IP) formation. In contrast, PMA inhibited these functions. The cells expressed conventional PKCs (, βI) and novel PKCs (δ, ε), and the effects of PKC inhibitors on NT binding were blocked by PKC downregulation. The inhibition of NT binding by PMA was enhanced by okadaic acid and blocked by PKC inhibitors. However, when some PKC inhibitors (rottlerin, BIS-1, Ro-318220, Go-69830, quercetin) were used at higher concentrations (> 2 μM), they had a different effect characterized by a dramatic increase in NT binding and an inhibition of NT-induced IP formation. The specificity of the agents implicated novel PKCs in this response and indeed, the inhibition of NT-induced IP formation was reproduced by PKCδ or PKCε knockdown. The inhibition of IP formation appeared to be specific to NT since it was not observed in response to bombesin. Scatchard analyses indicated that the PKC-directed agents modulated NT receptor affinity, not receptor number or receptor internalization. These findings suggest that PKC participates in heterologous regulation of NT receptor function by two mechanisms: a) — conventional PKCs inhibit NT receptor binding and signaling; and b) — novel PKCs maintain the ability of NT to stimulate PLC. Since NT can activate PKC upon binding to its receptor, it is possible that NT receptor is also subject to homologous regulation by PKC.     

Okadaic acid inhibits insulin-induced glucose transport in fetal brown adipocytes in an Akt-independent and protein kinase C zeta-dependent manner

In the present study we have investigated the effect of increased serine/threonine phosphorylation of insulin receptor substrates-1 and -2 (IRS-1 and IRS-2) by okadaic acid pretreatment on brown adipocyte insulin signalling leading to glucose transport, an important metabolic effect of insulin in brown adipose tissue. Okadaic acid pretreatment before insulin stimulation decreased IRS-1 and IRS-2 tyrosine phosphorylation in parallel to a decrease in their sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobility. IRS-1/IRS-2-associated p85alpha and phosphatidylinositol (PI) 3-kinase enzymatic activity were partly reduced in brown adipocytes pretreated with okadaic acid upon stimulation with insulin. Furthermore, insulin-induced glucose uptake was totally abolished by the inhibitor in parallel with a total inhibition of insulin-induced protein kinase C (PKC) zeta activity. However, activation of Akt/PKB or p70 S6 kinase (p70(s6k)) by insulin remained unaltered. Our results suggest that downstream of PI 3-kinase, insulin signalling diverges into at least two independent pathways through Akt/PKB and PKC zeta, the PKC zeta pathway contributing to glucose transport induced by insulin in fetal brown adipocytes.     

Rapid modulation of homing receptors (gp90MEL-14) induced by activators of protein kinase C. Receptor shedding due to accelerated proteolytic cleavage at the cell surface

Lymphocyte entry into lymph nodes and Peyer's patches is initiated by the adhesion of the lymphocytes to specialized postcapillary high endothelial venules (HEV). The binding of lymphocytes to lymph node HEV is mediated by the cell surface receptor gp90MEL-14 (gp90). Previous work has shown that gp90 is down-regulated over a period of days after mitogenic or mixed lymphocyte reaction stimulation of T lymphocytes. In our study, it is shown that stimulation of lymphocytes with activators of protein kinase C (PKC), such as PMA or 1-oleoyl 2-acetyl-glycerol, results in the nearly complete loss of surface expression of gp90 within 1 h. Pretreatment of the cells with H-7 or staurosporine, PKC inhibitors, but not HA1004, a general protein kinase inhibitor, prevents the loss of gp90MEL-14. Within 15 min of stimulation of PKC, a novel form of gp90 can be immunoprecipitated from the supernatant of stimulated cells. Upon deglycosylation, this soluble gp90 polypeptide is shown to be 12 kDa smaller than the cell surface protein. Peptide mapping showed identical patterns for surface and soluble receptor, confirming that the soluble Ag is related to the cell membrane protein. Together, these experiments suggest that activation of PKC results in the proteolytic cleavage of gp90MEL-14, resulting in receptor shedding and the inability of the lymphocytes to adhere to HEV endothelium. Furthermore, because supernatant from unstimulated, normal lymphocytes also contains a small amount of the low Mr form of gp90, cell surface proteolysis may be part of the normal turnover of this receptor glycoprotein. These experiments suggest that PKC may play a role in the regulation of lymphocyte traffic to lymphoid tissues.     

FKBP, the binding protein for the immunosuppressive drug, FK-506, is not an inhibitor of protein kinase C activity.

Recently, the amino acid sequence of a 12 Kd endogenous protein inhibitor of protein kinase C (PKC-I 2) has been shown to be identical to that of the 12 KDa receptor for the immunosuppressive drug, FK-506. In view of this observation we examined the effects of recombinant and native human FKBP on protein kinase C (PKC) activity. FKBP, at molar concentrations up to 1900-fold over that of PKC, failed to inhibit PKC phosphorylation of histone H1 and failed to block the auto-phosphorylation of PKC. Interestingly, FKBP is phosphorylated by PKC in these reactions. The phosphorylation of FKBP by PKC appears to be specific since the catalytic subunit of cAMP-dependent protein kinase fails to phosphorylate the binding protein. Our results fail to support a role for FKBP as an inhibitor of protein kinase C.     

Insulin action in vivo on a growth-related protein kinase in lactating mouse mammary gland

1. A cytosol serine-protein kinase was isolated and partially purified from lactating mouse mammary gland which is cAMP-independent, inhibited by the bioflavonoid quercetin and probably connected to the growth of mammary cells. 2. After supra-physiological doses of insulin (0.4 IU daily) given in vivo the activity of this kinase rises 2.4 times, but at 5 times higher doses of insulin the activity was completely inhibited. 3. The biphasic effect of insulin suggests that this protein kinase might be connected to the growth effect of the hormone.     

Protein tyrosine kinase Syk modulates EGFR signalling in human mammary epithelial cells

Signalling through protein tyrosine kinases (PTKs) is critical in the regulation of important cellular processes and its deregulation is associated with pathophysiological disorders such as cancer. We investigated the function of the PTK spleen tyrosine kinase (Syk) in the regulation of growth factor signalling pathways in human mammary epithelial cells. Our results show that downregulation of endogenous Syk expression enhances the ligand-induced activity of the epidermal growth factor receptor (EGFR) but not that of the closely related human epidermal growth factor receptor 2 (HER2) and human epidermal growth factor receptor 3 (HER3) receptors. Moreover, Syk function interfered with EGFR-mediated cell responses such as proliferation and survival of mammary epithelial cells. A mechanistic link between Syk and EGFR is further supported by the colocalisation of the two PTKs in membrane fractions as well as the regulatory feedback effects of the EGFR kinase on Syk activity. Our findings demonstrate that Syk acts a negative control element of EGFR signalling.     

Adaptive shifts in the activity of amino acid transport into mammary secretory cells in changed nutritional conditions

On the basis of analysis of published data, direct (using 13C) and indirect methods of estimating the amino acid transfer into mammary secretory cells in vivo were compared and the modified indirect method was used to determine quantitatively the shifts in transport activity in lactating cows and goats in trials with amino acid deficit or excess and in investigations that used a hyperinsulemic-euglycemic clamp. The analysis suggests that inadequacy of traditional use of extraction efficiency as a measure of tissue affinity to substrate is associated with two shortcomings: 1) if the changes in mammary blood flow are more expressed compared to transport activity, the arteriovenous difference and extraction efficiency may change in opposite direction to the shifts in transport activity; 2) due to the effect of nonlinearity, in situations characterized by small ratio of blood flow: the transport activity extraction efficiency is insensitive to shifts on activity of transport. The re-analysis of published data using the modified inbdirect method indicated that the deficit of individual amino acid caused a rise in activity of their transport and the excess decreased a net transfer into cell. The insulineuglycemic clamp treatments increased the activity of amino acid transport into the mammary cell and milk protein yield. The results obtained suggest that net transmembrane transfer of amino acid into mammary secretory cell can be controlled by the cell itself according to metabolic demand.     

Insulin and rabbit anti-insulin receptor antibodies stimulate additively the intrinsic receptor kinase activity.

This paper describes the properties of rabbit polyclonal antibodies directed against purified human insulin receptor which strongly stimulate the intrinsic tyrosine kinase activity. The stimulatory effect of the antibodies on the kinase activity was obtained on the insulin receptor autophosphorylation as well as on the kinase activity towards a synthetic substrate. This stimulation is additive to that induced by insulin. Moreover, rabbit antibodies do not impair insulin binding. These data strongly suggest that antibodies and insulin act through separate pathways. This conclusion is reinforced by the differences observed on the phosphopeptide maps of the receptor's beta subunit whose phosphorylation was performed either in the presence of insulin or rabbit antibodies. Interestingly, these polyclonal antibodies can also induce an activation of the receptor autophosphorylation by interacting only with extracellular determinants. The anti-insulin receptor antibodies mimic insulin in their stimulatory effect on amino acid (AIB) uptake, but they have a different effect to that found on the kinase activity; the simultaneous addition of the antiserum and insulin failed to stimulate this amino acid transport over the level induced by a saturating concentration of hormone.