Regulation of invasive behavior by vascular endothelial growth factor is HEF1-dependent.

We previously reported a vascular endothelial growth factor (VEGF) autocrine loop in head and neck squamous cell carcinoma (HNSCC) cell lines, supporting a role for VEGF in HNSCC tumorigenesis. Using a phosphotyrosine proteomics approach, we screened the HNSCC cell line, squamous cell carcinoma-9 for effectors of VEGFR2 signaling. A cluster of proteins involved in cell migration and invasion, including the p130Cas paralog, human enhancer of filamentation 1 (HEF1/Cas-L/Nedd9) was identified. HEF1 silencing and overexpression studies revealed a role for VEGF in regulating cell migration, invasion and matrix metalloproteinase (MMP) expression in a HEF1-dependent manner. Moreover, cells plated on extracellular matrix-coated coverslips showed enhanced invadopodia formation in response to VEGF that was HEF1-dependent. Immunolocalization revealed that HEF1 colocalized to invadopodia with MT1-MMP. Analysis of HNSCC tissue microarrays for HEF1 immunoreactivity revealed a 6.5-fold increase in the odds of having a metastasis with a high HEF1 score compared with a low HEF1 score. These findings suggest that HEF1 may be prognostic for advanced stage HNSCC. They also show for the first time that HEF1 is required for VEGF-mediated HNSCC cell migration and invasion, consistent with HEF1's recent identification as a metastatic regulator. These results support a strategy targeting VEGF:VEGFR2 in HNSCC therapeutics.

Synthesis and characterization of insulin-like growth factor (IGF)-1 photoprobes selective for the IGF-binding proteins (IGFBPS). photoaffinity labeling of the IGF-binding domain on IGFBP-2.

Elevated insulin-like growth factor (IGF)-1 levels are prognostic for the development of prostate and breast cancers and exacerbate the complications of diabetes. In each case, perturbation of the balance between IGF-1/2, the IGF-1 receptor, and the IGF-binding proteins (IGFBPs) leads to elevated IGF-1 sensitivity. Blockade of IGF action in these diseases would be clinically significant. Unfortunately, effective IGF antagonists are currently unavailable. The IGFBPs exhibit high affinity and specificity for the IGFs and serve as natural IGF antagonists, limiting their mitogenic/anti-apoptotic effects. As an initial step in designing IGFBP-based agents that antagonize IGF action, we have begun to analyze the structure of the IGF-binding site on IGFBP-2. To this end, two IGF-1 photoprobes, N(alphaGly1)-(4-azidobenzoyl)-IGF-1 (abG(1)IGF-1) and N(alphaGly1)-([2-6-(biotinamido)-2(p-azidobenzamido)hexanoamido]ethyl-1,3'-dithiopropionoyl)-IGF-1 (bedG(1)IGF-1), selective for the IGFBPs were synthesized by derivatization of the alpha-amino group of Gly(1), known to be part of the IGFBP-binding domain. Mass spectrometric analysis of the reduced, alkylated, and trypsin-digested abG(1)IGF-1.recombinant human IGFBP-2 (rhIGFBP-2) complex indicated photoincorporation near the carboxyl terminus of rhIGFBP-2, between residues 266 and 287. Mass spectrometric analysis of avidin-purified tryptic peptides of the bedG(1)IGF-1.rhIGFBP-2 complex revealed photoincorporation within residues 212-227. Taken together, these data indicate that the IGFBP-binding domain on IGF-1 contacts the distal third of IGFBP-2, providing evidence that the IGF-1-binding domain is located within the C terminus of IGFBP-2.

Elevated glucose increases mesangial cell sensitivity to insulin-like growth factor I.

To determine the effects of glucose on insulin-like growth factor I (IGF-I)-induced mesangial cell (MC) proliferation, we have examined the relationships between IGF binding protein 2 (IGFBP-2) secretion and proliferation in murine MCs (MMCs). MMCs incubated in high glucose (HG, 25 mM) exhibited a 25-30% reduction in IGFBP-2 secretion compared with cells in normal glucose (NG, 5.6 mM). This loss was not due to cell surface binding; it correlated with a 3.1-fold decrease in IGFBP-2 mRNA. IGFBP-2 secretion was stimulated by IGF-I in NG but was unaltered in HG. Insulin treatment yielded similar results at 10-fold higher doses, indicating that this response is IGF-I receptor dependent. MMCs in HG displayed increased IGF-I-stimulated insulin receptor substrate-1/2 phosphorylation and activator protein-1 transcriptional activity compared with NG controls. Accordingly, although IGF-I was not proliferative in NG, it increased [3H]thymidine incorporation and cell number in HG to an extent proportional to the decrease in IGFBP-2. Thus hyperglycemia, as seen in diabetes, may increase MC IGF-I sensitivity by reducing IGFBP-2 expression, in turn increasing its proliferative and secretory responses and contributing to the development of diabetic glomerulosclerosis.

Oleic acid and angiotensin II induce a synergistic mitogenic response in vascular smooth muscle cells.

Oleic acid and angiotensin II (Ang II) are elevated and may interact to accelerate vascular disease in obese hypertensive patients. We studied the effects of oleic acid and Ang II on growth responses of rat aortic smooth muscle cells (VSMCs). Oleic acid (50 micromol/L) raised thymidine incorporation by 50% at 24 hours and cell number by 55% at 6 days (P<.05). Ang II (10(-11) to 10(-6) mol/L) did not significantly increase thymidine incorporation or VSMC number. Combining Ang II and 50 micromol/L oleic acid doubled thymidine incorporation and VSMC number. Losartan, an angiotensin type 1 (AT1) receptor antagonist, blocked the synergistic interaction between Ang II and oleic acid, whereas the AT2 receptor antagonist PD 123319 did not. Protein kinase C inhibition and downregulation, as well as inhibition of extracellular signal-regulated kinase (ERK) activation by PD 98059, eliminated the rise of thymidine incorporation in response to oleic acid and the synergistic interaction with Ang II. However, the response to 10% fetal bovine serum was unaffected. An antisense oligodeoxynucleotide to ERK-1 and ERK-2 reduced ERK protein expression and activation by 83% and 75%, respectively. Antisense prevented the rise of thymidine incorporation in response to oleic acid and the synergy with Ang II. Antisense reduced but did not prevent increased thymidine incorporation in response to serum. The data indicate that oleic acid and Ang II exert a synergistic mitogenic effect in VSMCs and suggest an important role for the AT1 receptor, PKC, and ERK in this synergy. The observations raise the possibility that a synergistic mitogenic interaction between oleic acid and Ang II accelerates vascular remodeling in obese hypertensive patients.

Bradykinin induces tubulin phosphorylation and nuclear translocation of MAP kinase in mesangial cells.

Glomerular hypertension and glomerular hypertrophy act early and synergistically to promote glomerular injury in diabetes. We have previously shown that increased renal kinin production contributes to the glomerular hemodynamic abnormalities associated with diabetes. Glomerulosclerosis, characterized by mesangial cell proliferation and matrix expansion, is the final pathway leading to renal failure. The signal(s) initiating mesangial cell proliferation is ill defined. In the present study, we utilized immunofluorescence, immunoprecipitation, and immunoblotting techniques to identify substrates that are tyrosine phosphorylated in response to bradykinin action in mesangial cells. Immunofluorescence microscopy of mesangial cells stained with anti-phosphotyrosine (anti-PY) antibodies following bradykinin treatment (10(-9)-10(-6) M) revealed a dose-dependent increase in the labeling of cytoplasmic and nuclear proteins. Immunoprecipitation with anti-PY, followed by immunoblot revealed bradykinin-induced tyrosyl phosphorylation of tubulin and mitogen-activated protein kinase (MAPK). Confocal microscopy of mesangial cells stained for MAPK indicated that bradykinin stimulation resulted in translocation of MAPK from the cytoplasm to the nucleus by 2 h. These data demonstrate that bradykinin action results in the tyrosine phosphorylation of cellular proteins in mesangial cells and suggest a role for tubulin and MAPK in the signaling cascade of bradykinin leading to altered mesangial function.

Activation of C-jun N-terminal kinase/stress-activated protein kinase in primary glial cultures.

Glial cells in the mammalian CNS are subject to environmental stress resulting from a variety of neuro-pathological conditions. In this study, we have examined the activation of a stress signal responsive kinase, i.e., stress-activated protein kinase (SAPK) or c-Jun N-terminal kinase (JNK), in primary cultures of rat brain glial cells (i.e., astrocytes and oligodendrocytes) and an oligodendrocyte progenitor cell line, CG4, in response to cytokines and other stress inducers. JNK/SAPK activity was measured by an immune complex kinase assay using polyclonal anti-JNK antibodies along with GST c-Jun (1-79) as the substrate. Among the cytokines tested, TNF-alpha had the strongest effect on JNK activation followed by TNF-beta in both the glial cell types while a substantial level of kinase activation was observed in response to IL-1 in astrocytes. JNK activation by TNF-alpha in astrocytes, but not in oligodendrocytes, showed a biphasic response. An in-gel kinase assay of cell extracts and immunoprecipitated JNK confirmed the activation of JNK1 in cells treated with TNF-alpha. JNK was also activated by several other stress-inducing factors including. UV light, heat shock, inhibitors of protein synthesis, and mechanical injury. Incubation of cells with bacterial sphingomyelinase and a cell-permeable ceramide stimulated JNK activity, suggesting that the ceramide pathway may play a role in JNK activation, although the time course of activation did not correspond to that of TNF-alpha. The results are discussed in terms of possible roles of JNK activation in signaling for gliosis in astrocytes and as a protective/toxic response in oligodendrocytes.

Oleic acid-induced mitogenic signaling in vascular smooth muscle cells. A role for protein kinase C.

As an initial step in testing the hypothesis that high oleic acid concentrations contribute to vascular remodeling in obese hypertensive patients by activating protein kinase C (PKC), the effects of oleic acid on primary cultures of rat aortic smooth muscle cells (RASMCs) were studied. Oleic acid, an 18-carbon cis-monounsaturated fatty acid (18:1 [cis]), from 25 to 200 mumol/L significantly increased [3H]thymidine uptake in RASMCs with an EC50 of 41.0 mumol/L and a maximal response of 196 +/- 15% of control (P < .01). Oleic acid from 25 to 200 mumol/L caused a concentration-dependent increase in the number of RASMCs in culture at 6 days, reaching a maximum of 210 +/- 13% of control at 100 mumol/L (P < .001). PKC inhibition with 4 mumol/L bisindolyImaleimide I and PKC depletion (alpha, mu, iota, and zeta) with 24-hour exposure to 200 nmol/L phorbol 12-myristate 13-acetate in RASMCs eliminated the mitogenic effects of oleic acid but did not reduce responses to 10% FBS. Stimulation of intact cells with oleic acid induced a peak increase of cytosolic PKC activity, reaching 328 +/- 8% of control (P < .001), but did not enhance PKC activity in the membrane fraction (105 +/- 4%, P = NS). The oleic acid-induced increase of PKC activity in cell lysates was similar in the presence and absence of Ca2+, phosphatidylserine, and diolein (maximum response, 360 +/- 4% versus 342 +/- 9% of control, P = NS). Unlike phorbol 12-myristate 13-acetate, oleic acid over 24 hours did not downregulate any of the four PKC isoforms detected in RASMCs. Oleic acid treatment activated mitogen-activated protein (MAP) kinase. PKC depletion in RASMCs eliminated the rise in thymidine uptake, activation of PKC, and activation of MAP kinase in response to oleic acid. In contrast to oleic acid, 50 to 200 mumol/L stearic (18:0) and elaidic (18:1 [trans]) acids, which are less effective activators of PKC than oleic acid, did not enhance thymidine uptake. These data suggest that oleic acid induces proliferation of RASMCs by activating PKC, particularly one or more of the Ca(2+)-independent isoforms, and raise the possibility that the higher oleic acid concentrations observed in obese hypertensive patients may contribute to vascular remodeling.

Activation of cJun NH2-terminal kinase/stress-activated protein kinase by insulin.

One of insulin's many biological effects is the increased transcription of AP-1-regulated genes. cJun is the principal component of the AP-1 transcription complex, which is regulated by the newly discovered members of the MAPK superfamily referred to as cJun NH2-terminal kinases (JNKs) or stress-activated protein kinases (SAPKs). We show that insulin stimulates a dose- and time-dependent increase in JNK activity in Rat 1 fibroblasts overexpressing human insulin receptors (Rat 1 HIR cells). Using two different polyclonal anti-JNK antibodies, JNK activity was measured after immunoprecipitation from whole cell extracts by phosphorylation of GSTcJun(1-79). Peak activation occurred 15 min after insulin addition, resulting in a 2.5-fold increase in GSTcJun(1-79) phosphorylation over unstimulated controls. Maximal JNK activation correlated with the onset of AP-1 DNA binding activity. Both insulin-stimulated JNK activity and insulin-induced AP-1 transcriptional activity were found to be Ras-dependent. These data suggest that in Rat 1 cells, JNK activation may play a role in insulin-regulated AP-1 transcriptional activity leading to a mitogenic response.

Neuronal insulin receptors in Y79 retinoblastoma cells.

Immunoblot analysis of Y79 cell membrane proteins indicated that Y79 insulin receptors (InsRs) alpha subunits had a mass of 115 kDa. Biosynthetic studies revealed a typical transit time for InsR delivery to the Golgi (approximately 2h) and receptor processing. However, neither the proreceptor nor the mature receptor exhibited endoglycosidase H-resistance, consistent with a lack of N-linked glycan processing. Insulin stimulated a rapid and transient tyrosine phosphorylation of receptor beta subunits (95 kDa) and of IRS-1 in intact Y79 cells, whereas in vitro studies with enriched membrane glycoproteins resulted in the autophosphorylation of both InsR (95 kDa) and IGF-1-R (98k Da) beta subunits. These studies provide the first biochemical dissection of InsR structure and function in retinoblastoma cells.

Characterization of vasoactive intestinal peptide receptors in rabbit ciliary processes.

PURPOSE: To demonstrate a potential role for vasoactive intestinal peptide (VIP) in the regulation of ciliary process function, VIP receptors on rabbit ciliary process membranes were identified and characterized in biochemical and immunochemical studies. METHODS: Membranes were isolated from rabbit ciliary processes, and VIP receptors were characterized by competition binding, affinity cross-linking, and N-glycanase digestion. A site-specific polyclonal antibody directed against the NH2-terminal end of the deduced sequence of the recently cloned rat VIP receptor was generated and used to identify the VIP receptor by immunoblot analysis. RESULTS: Membranes isolated from rabbit ciliary processes exhibited a high-affinity VIP binding site (KD approximately 1 nM). Secretin and glucagon, which possess considerable primary sequence homology with VIP, were ineffective in inhibiting 125I-VIP binding to ciliary process membranes. In conjunction with the chemical cross-linking agent disuccinimidyl suberate, 125I-VIP specifically labeled a 63-kd protein in membranes from ciliary processes. This apparent size was confirmed by immunoblot analysis of ciliary body membranes using a site-specific polyclonal antibody that recognizes residues 92 to 104 of the rat VIP receptor. Digestion of the affinity-labeled receptor with N-glycanase generated an N-linked oligosaccharide free core protein of -50 kd. CONCLUSIONS: These findings demonstrate the presence of specific VIP receptors in rabbit ciliary processes. The differences in ligand specificity and structure of the ciliary process VIP receptor, compared to VIP receptors on peripheral tissues, suggest either a specific role(s) for VIP that may be unique to the anterior segment or the existence of VIP receptor isoforms.

Characterization of the G-protein linked orphan receptor GPRN1/RDC1.

Site specific antibodies were raised against the second intracellular loop of the G-protein coupled orphan receptor GPRN1 for analysis of receptor protein expression from a number of sources. Immunoblot analyses showed GPRN1 to be a 60 kDa membrane bound glycoprotein. It was found in AR4-2J, PC12 and SK-N-MC cell lines. High amounts of GPRN1 were found in rat stomach, liver, lung, brain, small intestine and pancreas, with lower amounts in spleen and kidney: none was detectable in rat heart. This distribution differs markedly from that of the closely related orphan receptor RDC1. The receptor shows high evolutionary conservation: immunoreactive 60 kDa GPRN1 was detected in membrane glycoprotein fractions from the livers of rat, toad, chick, stingray and shark. It is therefore predicted that GPRN1 and RDC1 represent two tissue specific subtypes of a 60 kDa receptor for a ligand which has likely been highly conserved throughout evolution and which is active upon many organs of the body.

Specific recognition of the human neuroendocrine receptor for vasoactive intestinal peptide by anti-peptide antibodies.

Rabbit polyclonal IgG antibodies were generated to three distinct synthetic peptide substituents of the human neuroendocrine-type 7 transmembrane-domain receptor for vasoactive intestinal peptide (VIP), including a portion of the amino-terminus, first extracellular loop, and carboxyl-terminus. Immunofluorescent staining of both human K293 cell transfectants, expressing recombinant VIP receptors, and HT-29 human intestinal epithelial cells, bearing native VIP receptors, was observed with each of the antibodies and was eliminated specifically after absorption of antibodies with the respective peptide immunogen. Each of the antibodies recognized the same approximately 70-kDa membrane proteins, extracted from both K293 cell transfectants and HT-29 cells, in sodium dodecyl sulfate-polyacrylamide gel electrophoresis blots. Neither IgG nor Fab preparations of the antibodies inhibited VIP binding to cellular receptors at a concentration of 1 microgram/ml, that yielded optimal immunofluorescence, or at 5-300 micrograms/ml. In contrast, 5-200 micrograms/ml of anti-peptide antibodies as IgG, but not Fab, significantly inhibited the increase in concentration of cyclic AMP in HT-29 cells elicited by 1 nM VIP, without affecting the greater increase evoked by 100 nM VIP or alone altering the level of cyclic AMP. Antibodies to several peptide substituents thus bind specifically to VIP receptors in immunoblots and permeabilized cells, and may affect the cellular functions of VIP receptors with sufficient selectivity to reduce transduction of signals, without altering the binding of VIP.

Insulin like growth factor 1 receptor signal transduction to the nucleus.

The mechanism by which IGF-1Rs regulate the growth and maintenance of cells in normal and disease states provides an important setting for studies addressing signal transduction events at the nuclear level. With the identification of c-Jun/AP-1 as a nuclear target of IGF-1 action we are provided with a model system for pursuing the molecular mechanisms triggered by IGF-1 action.

Differential expression of retinal insulin receptors in STZ-induced diabetic rats.

A major complication of diabetes mellitus is retinopathy, which is characterized by increased neovascularization and neuronal degeneration in the retina. The biochemical processes underlying these changes are largely unknown. To better understand the role(s) of insulin or its lack and the resultant hyperglycemia in the etiology of these events, peripheral and neuronal (having 125 kDa and 115 kDa alpha subunits, respectively) insulin receptor subtype levels in the retinas of Streptozocin-induced diabetic rats were quantified. Immunoblot analysis of wheat germ agglutinin-agarose purified retinal membrane proteins revealed that retinas from diabetic rats expressed higher insulin receptor levels than retinas from control rats. This increase reflected a doubling of neuronal and a approximately 20% decrease in peripheral insulin receptor subtypes, respectively. Insulin-treated diabetic rats had neuronal receptor levels equal to control values, at the same time having a further reduced number of peripheral insulin receptors relative to controls. Affinity labeling analysis of WGA-purified retinal membrane proteins indicated a 1.5-fold increase in neuronal and a 9% decrease in peripheral receptor subtypes, corroborating the immunoblot analysis. Neuronal insulin receptors in WGA-purified cortical synaptosomal membranes also were increased in diabetic rats, with insulin treatment reducing this effect. The up-regulated receptors retained their ability to undergo insulin-dependent autophosphorylation and, as such, did not appear functionally impaired. These data suggest that the expression of neuronal insulin receptors in retina and brain and peripheral insulin receptors in the retina of diabetic rats is sensitive to levels of insulin/glucose in peripheral circulation.

Multinucleated rat alveolar macrophages express functional receptors for calcitonin.

The fusion of mononuclear phagocytes occurs spontaneously in vivo and leads to the differentiation of either multinucleated giant cells or osteoclasts in chronic inflammatory sites or in bone, respectively. Although osteoclasts are responsible for resorbing bone, the functional role of giant cells in chronic inflammatory reactions and tumors remains poorly understood. We recently reported that the plasma membrane of multinucleated macrophages is, like that of osteoclasts, enriched in Na-K-adenosinetriphosphatases (ATPases). We also observed that the localization of their Na-K-ATPases is restricted to the nonadherent domain of the plasma membrane of cells both in vivo and in vitro, thus imposing a functional polarity on their organization. By following this observation, we wished to investigate whether these cells also expressed, like osteoclasts, functional receptors for calcitonin (CT). To this end, alveolar macrophages were fused in vitro, and both their structural and functional association with CT was analyzed and compared with those of mononucleated peritoneal and alveolar macrophages. Evidence is presented that multinucleated alveolar macrophages express a high copy number of functional receptors for CT. Our results also indicate that alveolar macrophages, much like peritoneal, express functional receptors for calcitonin gene-related peptide. It is suggested that multinucleated rat alveolar macrophages offer a novel model system to study CT receptors and that calcitonin may control local immune reactions where giant cells differentiate.

Insulin-like growth factor-1 induces tyrosyl phosphorylation of nuclear proteins.

Stimulation of mesangial cells with insulin-like growth factor-1 (IGF-1) resulted in the rapid tyrosyl phosphorylation of nuclear proteins as indicated by fluorescence microscopy of cells stained with anti-phosphotyrosine antibodies. Immunoprecipitation of nuclear extracts with anti-phosphotyrosine antibodies revealed that IGF-1 induced a transient increase in immunoreactive phosphotyrosine in nuclear proteins of 43, 95, and 160 kDa. Using a double immunoprecipitation protocol, the transcription factor c-Jun was also found to increase in immunoreactive phosphotyrosine in response to IGF-1. A similar pattern of tyrosyl phosphorylation of nuclear proteins was observed in the epidermoid carcinoma cell line CaSki. These data suggest that tyrosyl phosphorylation of nuclear proteins may be a step in the transduction of mitogenic signals.

Increased hepatic insulin proreceptor-to-receptor ratio in diabetes: a possible processing defect.

Hepatic insulin proreceptors and receptors were studied in control and in ketotic diabetic rats 2-4 wk after streptozotocin treatment. Solubilized preparations were partially purified by wheat germ agglutinin-agarose (WGA) and lentil lectin agarose (LLA) chromatography to enrich eluates in insulin receptors and proreceptors, respectively. After phosphorylation with [gamma-32P]ATP, an approximately 190-kDa glycoprotein was identified in LLA eluates as the insulin proreceptor, based on insulin dose-dependent tyrosine autophosphorylation, immunoprecipitation with insulin receptor-specific antibodies, and high-mannose glycosylation. Mature approximately 95 kDa phosphorylated beta-subunits were present in both LLA and WGA eluates. LLA also showed phosphorylated partially processed beta-subunits (approximately 85 kDa) and proreceptors (approximately 190 kDa). Proreceptors comprised less than 1% of the total yield of hepatic insulin receptors. The incorporation of 32P into proreceptors (per gram liver or DNA) was 4.7- or 4.5-fold greater in diabetic vs. control rats, whereas receptor labeling increased only 1.8- or 1.5-fold in diabetic rats. beta-Subunit autophosphorylation per receptor was identical in control and diabetic rats. The phosphorylation data suggested a diabetes-associated 2.6-fold increase in proreceptor-to-receptor ratios. When assessed by cross-linking with 125I-labeled insulin or by immunoblotting, proreceptor-to-receptor ratios were increased 1.5- and 3.1-fold, respectively, in diabetic rats. The data suggest that uncontrolled diabetes may alter insulin receptor processing.

Characterization of a novel receptor in toad retina with dual specificity for insulin and insulin-like growth factor I.

The biochemical properties of insulin receptors from toad retinal membranes were examined in an effort to gain insight into the role this receptor plays in the retina. Competition binding assays revealed that toad retinal membranes contained binding sites that displayed an equal affinity for insulin and insulin-like growth factor I (IGF-I). Affinity labeling of toad retinal membrane proteins with 125I-insulin resulted in the specific labeling of insulin receptor alpha-subunits of approximately 105 kDa. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of partially reduced (alpha beta-heterodimer) receptors affinity-labeled with 125I-insulin indicated the presence of a disulfide-linked beta-subunit of approximately 95 kDa. Endoglycosidase F digestion of the affinity-labeled alpha-subunits increased their mobility by reducing their apparent mass to approximately 83 kDa. This receptor was not detected by immunoblot analysis with a site-specific antipeptide antibody directed against residues 657-670 of the carboxy terminal of the human insulin receptor alpha-subunit, whereas this antibody did label insulin receptor alpha-subunits from pig, cow, rabbit, and chick retinas. In in vitro autophosphorylation assays insulin stimulated the tyrosine phosphorylation of toad retina insulin receptor beta-subunits. These data indicate that toad retinal insulin receptors have a heterotetrameric structure whose alpha-subunits are smaller than other previously reported neuronal insulin receptors. They further suggest that a single receptor may account for both the insulin and IGF-I binding activities associated with toad retinal membranes.

Vasoactive intestinal peptide receptors on AR42J rat pancreatic acinar cells.

VIP receptors on AR42J rat pancreatic cells were analyzed by competition binding, affinity labeling and by N-glycanase digestion analyses. These studies revealed the presence of specific, high affinity (Kd approximately 1 nM) VIP receptors with a mass of 67 kDa or 59 kDa under reducing or non-reducing conditions, respectively. N-glycanase digestion of affinity labeled membranes generated a core receptor protein of approximately 44 kDa and evidence for at least two N-linked glycans on the mature receptor. The receptor lacked O-linked oligosaccharides but contained terminal sialic acid residues on its N-linked glycan(s) based on digestions with O-glycanase and neuraminidase. The similarity of the AR42J VIP receptor to the recently cloned cDNA for human VIP receptors makes this cell line an attractive model for further analysis of VIP receptor signal transduction events.

Regulation of insulin-like growth factor I receptors in diabetic mesangial cells.

Mesangial cells are thought to play a central role in the renal complications of diabetes mellitus. Insulin-like growth factor I (IGF-I) has been found to promote mesangial cell proliferation and regulate normal mesangial cell function in an autocrine and/or paracrine fashion. To gain further insight into the potential regulatory role IGF-I may play in mesangial cell function in diabetes, IGF-I receptors were analyzed in mesangial cells isolated from diabetic mice (db/db) and their control littermates (db/m). Mesangial cells isolated from db/db mice exhibited higher levels of IGF-I receptors compared to cells from db/m mice. Insulin receptors were not detectable in either cell type by binding analyses; however, immunoblot analysis revealed insulin receptor alpha-subunits in wheat germ agglutinin-Sepharose-purified membranes from db/db cells. Northern blot analysis further indicated a lack of detectable insulin receptor mRNA in db/m cells, whereas db/db cells expressed multiple insulin receptor mRNA transcripts. Both IGF-I and insulin receptor mRNA levels were increased in db/db cells grown in the presence of high glucose (28 mM), whereas the receptor protein levels remained relatively constant or increased, respectively. This increased expression of IGF-I and insulin receptors in diabetic mesangial cells may have an important role in the development of diabetic nephropathy.