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Biomedical subjects

R Lammers

Publications and source records attributed to R Lammers.

At least 19 recordsLinked to original sources
PubMed2005

A novel functional polymorphism (-336A/G) in the promoter of the partitioning-defective protein-6alpha gene is associated with increased glucose tolerance and lower concentrations of serum non-esterified fatty acids.

AIMS/HYPOTHESIS: Partitioning-defective protein-6alpha (Par6alpha) has recently been demonstrated to negatively regulate insulin signalling in murine myoblasts. To address whether Par6alpha plays a role in human physiology, the present study investigated whether mutations exist in the Par6alpha gene and whether these mutations, if present, are associated with pre-diabetic phenotypes in non-diabetic subjects. METHODS: The complete gene (part of the promoter [2.1 kb], all exons/introns and the 3' untranslated region) encoding Par6alpha was analysed in 664 non-diabetic subjects. We investigated possible associations between single nucleotide polymorphisms and percentage of body fat, glucose tolerance (as determined by OGTT), serum NEFA concentrations and whole-body insulin sensitivity (estimated during the OGTT, and for a subgroup of 242 subjects determined by the euglycaemic-hyperinsulinaemic clamp). RESULTS: A rare A/G polymorphism was found 336-bp upstream of the translational start codon (allele frequency 0.03). The data for subjects homozygous and heterozygous for -336G (R/G, n=43) were combined and compared with those for subjects homozygous for -336A (A/A, n=621). Subjects with the R/G genotype had lower fasting (4.84+/-0.09 mmol/l, means+/-SEM, p=0.049) and 2-h (5.50+/-0.02 mmol/l, p=0.050) plasma glucose concentrations than subjects with the A/A genotype (5.02+/-0.02 and 5.94+/-0.06 mmol/l, respectively). Subjects with the R/G genotype also had lower fasting (448+/-31 micromol/l, p=0.018) and 2-h serum NEFA concentrations (61+/-7 micromol/l, p=0.015) than subjects with the A/A genotype (529+/-9 and 75+/-2 micromol/l, respectively), adjusted for age, sex and percentage of body fat. There were no differences in adiposity or whole-body insulin sensitivity between the two genotype groups (all p>0.36). A luciferase reporter gene assay revealed that the -336G promoter variant had a significantly lower (-22.8%, p=0.006) transcriptional activity in transfected C2C12 murine myoblasts than the -336A promoter variant. CONCLUSIONS/INTERPRETATION: A novel functional variant in the promoter of the Par6alpha gene is associated with reduced fasting glycaemia, increased glucose tolerance and reduced serum NEFA concentrations.

Adult↗
PubMed2003

Induction of adiponectin gene expression in human myotubes by an adiponectin-containing HEK293 cell culture supernatant.

AIMS/HYPOTHESIS: Adiponectin, an adipocytokine known to be down-regulated in obesity-linked disorders, is considered to be a potential key mediator of insulin sensitivity. In this study, we asked whether adiponectin is able to regulate ten selected genes possibly associated with insulin sensitivity in human skeletal muscle cells. METHODS: To this end, we treated in vitro differentiated human myotubes with the culture supernatant of HEK293 cells stably transfected with human recombinant adiponectin and assessed gene expression by RT-PCR. Intracellular adiponectin protein was quantified by radioimmunoassay and visualized by Western blotting. RESULTS: In contrast to the control supernatant, the adiponectin-containing supernatant consistently induced expression of adiponectin mRNA in human myotubes from eight different donors (mean increase: 90-fold over control; n=8, p<0.001). This increase in mRNA was paralleled by a rise in intracellular adiponectin protein (mean increase: 8.3-fold over control; n=4, p<0.05). Expression of the other nine candidate genes was not altered. In human skin fibroblasts and HepG2 cells, the adiponectin-enriched supernatant did not induce relevant amounts of adiponectin mRNA. CONCLUSIONS/INTERPRETATION: In conclusion, we show here that adiponectin gene expression is specifically inducible in skeletal muscle cells.

Adiponectin↗
PubMed2002

Polymorphism of the CNS active drug Org 13011: the application of high temperature analysis to detect new polymorphs.

The polymorphism of the CNS active compound Org 13011 was studied using different crystallisation methods (i.e. different solvents and cooling rates). The samples were analysed by Raman, solid state NMR, X-ray powder diffraction (XRPD) and thermal analysis. This led to the characterisation of two crystalline forms A and B. Further high temperature analysis using Raman, XRPD, solid state NMR and DSC revealed another two (high temperature) crystalline forms C and D. The transitions to the high temperature crystalline forms occur at temperatures of about 60 degrees C. This study shows that the application of high temperature experiments is useful and can lead to the discovery of new crystalline forms.

Central Nervous System Agents↗
PubMed2001

Comparative study of protein tyrosine phosphatase-epsilon isoforms: membrane localization confers specificity in cellular signalling.

To study the influence of subcellular localization as a determinant of signal transduction specificity, we assessed the effects of wild-type transmembrane and cytoplasmic protein tyrosine phosphatase (PTP) epsilon on tyrosine kinase signalling in baby hamster kidney (BHK) cells overexpressing the insulin receptor (BHK-IR). The efficiency by which differently localized PTPepsilon and PTPalpha variants attenuated insulin-induced cell rounding and detachment was determined in a functional clonal-selection assay and in stable cell lines. Compared with the corresponding receptor-type PTPs, the cytoplasmic PTPs (cytPTPs) were considerably less efficient in generating insulin-resistant clones, and exceptionally high compensatory expression levels were required to counteract phosphotyrosine-based signal transduction. Targeting of cytPTPepsilon to the plasma membrane via the Lck-tyrosine kinase dual acylation motif restored high rescue efficiency and abolished the need for high cytPTPepsilon levels. Consistent with these results, expression levels and subcellular localization of PTPepsilon were also found to determine the phosphorylation level of cellular proteins including focal adhesion kinase (FAK). Furthermore, PTPepsilon stabilized binding of phosphorylated FAK to Src, suggesting this complex as a possible mediator of the PTPepsilon inhibitory response to insulin-induced cell rounding and detachment in BHK-IR cells. Taken together, the present localization-function study indicates that transcriptional control of the subcellular localization of PTPepsilon may provide a molecular mechanism that determines PTPepsilon substrate selectivity and isoform-specific function.

Animals↗
PubMed2001

Insulin inhibits leptin receptor signalling in HEK293 cells at the level of janus kinase-2: a potential mechanism for hyperinsulinaemia-associated leptin resistance.

AIMS/HYPOTHESIS: Leptin resistance in obese humans seems to be predominantly caused by signalling abnormalities at the post receptor level. Leptin resistance in obese individuals is frequently associated with insulin resistance and pronounced hyperinsulinaemia indicating a negative crosstalk of the insulin and leptin signalling chain. METHODS: This hypothesis was tested using a cell model of peripheral leptin signalling, i. e. insulin-secreting cell lines (RINr1046-38). Mechanisms for a crosstalk between the insulin and leptin signalling pathway were also studied in rat-1 and HEK293 cells overexpressing elements of the insulin and leptin signalling chain. RESULTS: The effects of leptin on insulin secretion are completely cancelled by a 4-h preincubation with 1 nmol/l insulin, supporting the hypothesis of a negative crosstalk of insulin and leptin signalling. We investigated the potential molecular mechanisms in more detail in HEK293 cells and Rat-1 fibroblasts that overexpressed proteins of the insulin and leptin signalling chain. Leptin (60 ng/ml) stimulated autophosphorylation of JAK-2 in HEK 293 cells. This leptin effect could be inhibited by simultaneous treatment of cells with insulin. Furthermore, overexpression of the insulin receptor in HEK 293 cells clearly reduced JAK-2 phosphorylation and led further downstream to a diminished phosphatidylinositol 3-kinase activity. The inhibitory effect of the insulin signal could be partially prevented by transfection of the cells with an inactive mutant of the tyrosine phosphatase SHP-1. CONCLUSION/INTERPRETATION: In summary, our data suggest that the insulin receptor signalling pathway interferes with leptin signalling at the level of JAK-2. Inhibition of JAK-2 phosphorylation might occur through SHP-1-dependent pathways, indicating that hyperinsulinaemia contributes to the pathogenesis of leptin resistance.

Animals↗
PubMed2001

Bacterial counts in experimental, contaminated crush wounds irrigated with various concentrations of cefazolin and penicillin.

The objective of this study was to determine if three different concentrations of cefazolin and penicillin irrigation solutions reduce quantitative bacterial counts in experimental crush wounds contaminated with multiple species of bacteria. The design used was a randomized, blinded, experimental animal study. An animal bite wound model was created by innoculating crushed incisions with three species of bacteria. Four paravertebral incisions extending to deep fascia were created in each of twelve anesthetized albino guinea pigs. Wound edges were clamped with a hemostat for five seconds to create crushed, devitalized tissue within each wound. Wounds were inoculated with 0.4 mL of a standard solution of Staphylococcus aureus, Bacterioides fragilis, and Pasturella multocida and covered. Four hours after inoculation, each wound was scrubbed for 30 seconds with 20% poloxamer 188 and then irrigated with 100 mL of one of four solutions: normal saline solution (control); cefazolin (CZ) 2 mg/mL, plus penicillin G (PCN) 200 units/mL (low dose); CZ 10 mg/mL, plus PCN 2,000 units/mL (intermediate dose); and CZ 50 mg/mL, plus PCN 20, 000 units/mL (high dose). Investigators were blinded to the solutions used. Wounds were covered with a vapor-permeable dressing. Six days after treatment, each wound was examined for signs of infection and then excised for quantitative bacteriologic analysis. Colony counts were reported as counts per gram of tissue. Wounds in the four irrigation solution groups were compared using ANOVA. A log difference of 3 was considered significant. The average log total bacteria/gram tissue for the four groups were: control, 4.35 (95% CI; 1.01); low dose, 4.09 (95% CI; 1.42); intermediate dose, 4.47 (95% CI; 1.27); and high dose, 3.45 (95% CI; 1.33). No wounds in the high-dose group had any clinical signs of infection, whereas 50% of wounds in the intermediate dose group, 42% in the low dose group, and 33% in the control group had either erythema, induration, or purulence. There were no statistically significant differences in the bacterial counts/gram tissue or clinical infection rates in any of the groups. A formal trend analysis failed to find a significant linear trend for decreasing bacterial counts for either antibiotic. In this experimental bite wound model containing contaminated, crushed tissue, irrigation with various solutions of cefazolin plus penicillin G did not reduce quantitative bacterial counts more than 3.1 log total bacteria/gram tissue.

Animals↗
PubMed2001

Inhibition of Ret oncogene activity by the protein tyrosine phosphatase SHP1.

Germline mutations in the Ret protooncogene give rise to the inherited endocrine cancer syndromes MEN types 2A and 2B and familiar medullary thyroid carcinoma. Although it is well accepted that the constitutive active tyrosine kinase of Ret oncogenes ultimately leads to malignant transformation, it is not clear whether a decrease in the autophosphorylation of oncogenic Ret forms can affect the mitogenic and transforming activities of Ret. Potential modulators of the tyrosine kinase activity of Ret could be tyrosine phosphatases that are expressed in human thyroid tissue. Therefore, we investigated the impact of the tyrosine phosphatases SHP1 and SHP2 on the intrinsic tyrosine kinase activity and oncogenic potency of Ret with a 9-bp duplication in the cysteine-rich domain (codons 634-636), which was described in a patient with MEN type 2A recently. SHP1 and SHP2 were stably overexpressed in NIH3T3 fibroblasts together with Ret-9bp. Coexpression of SHP1 with Ret-9bp reduced the autophosphorylation of Ret-9bp by 19 +/- 7% (P = 0.01, n = 4), whereas no effect was seen with SHP2. Furthermore, Ret-9bp could be coimmunoprecipitated with SHP1 but not with SHP2 antibodies. Suppression of the Ret-9bp tyrosine kinase activity by SHP1 caused a decrease in activation of Erk2 (extracellular signal-regulated kinase) and abolished PKB/Akt (protein kinase B) phosphorylation. In addition, diminished Ret-9bp autophosphorylation led to reduced phosphorylation of the transcription factor jun-D. Finally, the inhibitory effect on Ret-9bp signaling resulted in a 40-60% reduction of [(3)H]thymidine incorporation and in reduced ability of NIH3T3 cells to form colonies in soft agar. In conclusion, the data suggest that SHP1 caused a moderate reduction of Ret autophosphorylation, which led to a strong suppression of the Ret oncogene activity.

3T3 Cells↗
PubMed2000

Ret oncogene signal transduction via a IRS-2/PI 3-kinase/PKB and a SHC/Grb-2 dependent pathway: possible implication for transforming activity in NIH3T3 cells.

Multiple endocrine neoplasia 2A (MEN 2A) is an inherited disease caused by mutations of the Ret proto-oncogene. Although many different Ret mutations have been described, little is known about the signaling pathways triggered by the Ret oncogene. In this study, we have determined the signaling properties of a Ret-9bp duplication encoding amino acids 634-636, which was recently identified in a patient with all clinical features of the MEN 2A syndrome. The Ret-9bp duplication leads to constitutive activation of the Ret tyrosine kinase. Furthermore, Ret-9bp increased mitogenic and transforming activity demonstrated by thymidine incorporation as well as colony formation in soft agar. Studying intracellular signaling pathways, which may be involved in malignant transformation of Ret-9bp expressing NIH3T3 cells, we could demonstrate Ret-9bp dependent phosphorylation of insulin receptor substrate-2 (IRS-2) with consecutive activation of phosphatidylinositol 3-kinase (PI 3-kinase) and protein kinase B (PKB/AKT). Moreover, Ret-9bp induces phosphorylation of SHC resulting in growth factor receptor binding protein-2 (Grb-2) binding and activation of the mitogen activating protein (MAP) kinase pathway. In addition to these postreceptor cytoplasmic signaling events, we have studied nuclear signal by Ret-9bp and found activation of c-jun and jun-D, two members of the jun/AP-1 family of transcription factors. In summary, an oncogenic 9bp duplication of Ret causes Ret dimer formation and ligand independent activation of the tyrosine kinase. Besides the signaling steps leading to MAPK activation, we could demonstrate that Ret-9bp induced constitutive activation of a signaling pathway involving IRS-2, PI 3-kinase and PKB/AKT which could transduce the oncogenic Ret signal to increased gene transcription via activation of the jun/AP-1 transcription factor family.

3T3 Cells↗
PubMed2000

The carboxyl-terminal tyrosine residue of protein-tyrosine phosphatase alpha mediates association with focal adhesion plaques.

The receptor protein-tyrosine phosphatase alpha (PTPalpha) is involved in the activation of c-Src kinase as well as in down-regulation of the insulin signal. To investigate the role of PTPalpha in activation of the Src kinase in more detail we tried to overexpress this phosphatase in NIH3T3 fibroblasts. Although PTPalpha has been overexpressed in rat embryonic fibroblasts and in embryonic carcinoma cells and should increase mitogenic responses we were not able to achieve a detectable overexpression. In contrast, expression of partially (C442S) or completely inactive (C442S,C732S) PTPalpha or of phosphatase active PTPalpha containing mutation Y781F or Y798F was possible. The level of expression, however, was reduced to background after several passages of lines expressing PTPalphaC442S,C732S and PTPalphaY781F. When employed in a focus formation assay, only infection with virus encoding PTPalphaY798F induced Src-dependent formation of foci. In immunofluorescence studies, PTPalphaC442S and PTPalphaY781F but not PTPalphaY798F colocalized with proteins found in focal adhesion plaques. Treatment of PTPalphaC442S-overexpressing cells with vanadate abolished this colocalization and led to proteolytic processing of the phosphatase. We conclude that tyrosine 798 in PTPalpha is important for localization at focal adhesion plaques. Inhibition of phosphatases by vanadate treatment releases PTPalpha from focal adhesions.

3T3 Cells↗
PubMed2000

Serine residues 994 and 1023/25 are important for insulin receptor kinase inhibition by protein kinase C isoforms beta2 and theta.

AIMS/HYPOTHESIS: Inhibition of the signalling function of the human insulin receptor (HIR) is one of the principle mechanisms which induce cellular insulin resistance. It is speculated that serine residues in the insulin receptor beta-subunit are involved in receptor inhibition either as inhibitory phosphorylation sites or as part of receptor domains which bind inhibitory proteins or tyrosine phosphatases. As reported earlier we prepared 16 serine to alanine point mutations of the HIR and found that serine to alanine mutants HIR-994 and HIR-1023/25 showed increased tyrosine autophosphorylation when expressed in human embryonic kidney (HEK) 293 cells. In this study we examined whether these mutant receptors have a different susceptibility to inhibition by serine kinases or an altered tyrosine kinase activity. METHODS: Tyrosine kinase assay and transfection studies. RESULTS: In an in vitro kinase assay using IRS-1 as a substrate we could detect a higher intrinsic tyrosine kinase activity of both receptor constructs. Additionally, a higher capacity to phosphorylate the adapter protein Shc in intact cells was seen. To test the inhibition by serine kinases, the receptor constructs were expressed in HEK 293 cells together with IRS-1 and protein kinase C isoforms beta2 and theta. Phorbol ester stimulation of these cells reduced wild-type receptor autophosphorylation to 58 % or 55 % of the insulin simulated state, respectively. This inhibitory effect was not observed with HIR-994 and HIR-1023/25, although all other tested HIR mutants showed similar inhibition induced by protein kinase C. CONCLUSION/INTERPRETATION: The data suggest that the HIR-domain which contains the serine residues 994 and 1023/25 is important for the inhibitory effect of protein kinase C isoforms beta2 and theta on insulin receptor autophosphorylation.

Adenosine Triphosphate↗
PubMed2000

Serine residues 1177/78/82 of the insulin receptor are required for substrate phosphorylation but not autophosphorylation.

Serine residues of the human insulin receptor (HIR) may be phosphorylated and negatively regulate the insulin signal. We studied the impact of 16 serine residues in HIR by mutation to alanine and co-overexpression in human embryonic kidney (HEK) 293 cells together with the docking proteins insulin receptor substrate (IRS)-1, IRS-2, or (SHC) Src homologous and collagen-like. As a control, IRS-1 was also cotransfected with an HIR with a juxtamembrane deletion (HIR delta JM) and therefore not containing the domain required for interaction with IRS-1. Coexpression of HIR with IRS-1, IRS-2, and SHC strongly enhanced tyrosine phosphorylation of these proteins. A similar increase in tyrosine phosphorylation was observed in cells overexpressing IRS-1, IRS-2, or SHC together with all HIR mutants except HIR delta JM and a mutant carrying exchanges of serines 1177, 1178, and 1182 to alanine (HIR1177/78/82), although this mutant showed normal autophosphorylation. Analysis of total cell lysates with anti-phosphotyrosine antibodies showed that in addition to the overexpressed substrates, other cellular proteins displayed reduced levels of tyrosine phosphorylation in these cells. To study consequences for phosphatidylinositol 3-kinase (PI 3-kinase) activation, we established stable NIH3T3 fibroblast cell lines overexpressing wild-type HIR, HIR1177/78/82, and other HIR mutants as the control. Again, HIR1177/78/82 showed normal autophosphorylation but showed a clear decrease in tyrosine phosphorylation of endogenous IRS-1 and activation of PI 3-kinase. This decrease in kinase activity also occurred in an in vitro kinase assay towards recombinant IRS-1. Finally, we performed a separation of the phosphopeptides by high-performance liquid chromatography and could not detect any differences in the profiles of HIR and HIR1177/78/82. In conclusion, we have defined a region in HIR that is important for substrate phosphorylation but not autophosphorylation. Therefore, this mutant may provide new insights into the mechanism of kinase activation and substrate phosphorylation.

3T3 Cells↗
PubMed1999

The kinesin-like motor protein KIF1C occurs in intact cells as a dimer and associates with proteins of the 14-3-3 family.

Proteins of the kinesin superfamily are regulated in their motor activity as well as in their ability to bind to their cargo by carboxyl-terminal associating proteins and phosphorylation. KIF1C, a recently identified member of the KIF1/Unc104 family, was shown to be involved in the retrograde vesicle transport from the Golgi-apparatus to the endoplasmic reticulum. In a yeast two-hybrid screen using the carboxyl-terminal 350 amino acids of KIF1C as a bait, we identified as binding proteins 14-3-3 beta, gamma, epsilon, and zeta. In addition, a clone encoding the carboxyl-terminal 290 amino acids of KIF1C was found, indicating a potential for KIF1C to dimerize. Subsequent transient overexpression experiments showed that KIF1C can dimerize efficiently. However, in untransfected cells, only a small portion of KIF1C was detected as a dimer. The association of 14-3-3 proteins with KIF1C could be confirmed in transient expression systems and in untransfected cells and was dependent on the phosphorylation of serine 1092 located in a consensus binding sequence for 14-3-3 ligands. Serine 1092 was a substrate for the protein kinase casein kinase II in vitro, and inhibition of casein kinase II in cells diminished the association of KIF1C with 14-3-3gamma. Our data thus suggest that KIF1C can form dimers and is associated with proteins of the 14-3-3 family.

14-3-3 Proteins↗
PubMed1999

The inhibitory effect of 2-deoxyglucose on insulin receptor autophosphorylation does not depend on known serine phosphorylation sites or other conserved serine residues of the receptor beta-subunit.

Hyperglycemia induces insulin resistance in diabetic patients. It is known that supraphysiological levels of D-glucose or 2-deoxyglucose inhibit the insulin receptor and it is speculated that this effect is mediated by serine phosphorylation of the insulin receptor beta-subunit and other proteins of the insulin signaling chain. To test this hypothesis we prepared point mutations of the human insulin receptor where serine was exchanged to alanine at 16 different positions, either at known phosphorylation sites or at positions which are conserved in different tyrosine kinase receptors. These receptor constructs were expressed in HEK 293 cells and the effect of 2-deoxyglucose (25 mM) on insulin (100 nM) induced receptor autophosphorylation was studied. 2-Deoxyglucose consistently inhibits insulin stimulated autophosphorylation of all constructs to the same degree as observed in wild-type human insulin receptor. The data suggest that none of the chosen serine positions are involved in 2-deoxyglucose induced receptor inhibition.

Binding Sites↗
PubMed1998

Characterization of KIF1C, a new kinesin-like protein involved in vesicle transport from the Golgi apparatus to the endoplasmic reticulum.

Kinesins comprise a large family of microtubule-based motor proteins, of which individual members mediate specific types of motile processes. Using the ezrin domain of the protein-tyrosine phosphatase PTPD1 as a bait in a yeast two-hybrid screen, we identified a new kinesin-like protein, KIF1C. KIF1C represents a member of the Unc104 subfamily of kinesin-like proteins that are involved in the transport of mitochondria or synaptic vesicles in axons. Like its homologues, the 1103-amino acid protein KIF1C consists of an amino-terminal motor domain followed by a U104 domain and probably binds to target membranes through carboxyl-terminal sequences. Interestingly, KIF1C was tyrosine-phosphorylated after peroxovanadate stimulation when overexpressed in 293 or NIH3T3 fibroblasts or in native C2C12 cells. Using immunofluorescence, we found that KIF1C is localized primarily at the Golgi apparatus. In brefeldin A-treated cells, the Golgi membranes and KIF1C redistributed to the endoplasmic reticulum (ER). This brefeldin A-induced flow of Golgi membranes into the ER was inhibited in cells transiently overexpressing catalytically inactive KIF1C. In conclusion, our data suggest an involvement of tyrosine phosphorylation in the regulation of the Golgi to ER membrane flow and describe a new kinesin-like motor protein responsible for this transport.

Amino Acid Sequence↗
PubMed1998

Mutant forms of the protein tyrosine phosphatase alpha show differential activities towards intracellular substrates.

BHK cells overexpressing five million IR (BHK-IR) respond to insulin with reduced growth and detachment from the dish surface. We have recently identified protein tyrosine phosphatase (PTP) alpha as a negative regulator of the insulin receptor (IR) tyrosine kinase that is able to rescue BHK-IR cells from the insulin effect. In this report we describe the effect of several point mutations in PTP alpha on the phosphatase activity and regulation of insulin signaling in BHK-IR cells. Analysis of total cellular phosphotyrosine protein revealed several molecules that were dephosphorylated when PTP alpha or a phosphatase active mutant was overexpressed. By contrast, some proteins were tyrosine phosphorylated as strong or to an even higher extent as in the parental line when PTP alpha Y798F was present. We conclude that mutation of the carboxyterminal tyrosine in PTP alpha uncovers a dual function of this phosphatase in BHK cells: reduction of the IR signal and activation of an endogenous kinase.

Animals↗
PubMed1997

Intracellular signaling of the Ufo/Axl receptor tyrosine kinase is mediated mainly by a multi-substrate docking-site.

Ufo/Axl belongs to a new family of receptor tyrosine kinases with an extracellular structure similar to that of neural cell adhesion molecules. In order to elucidate intracellular signaling, the cytoplasmic moiety of Ufo/Axl was used to screen an expression library according to the CORT (cloning of receptor targets) method. Three putative Ufo substrates were identified: phospholipase Cgamma1 (PLCgamma), as well as p85alpha and p85beta subunits of phosphatidylinositol 3'-kinase (PI3-kinase). Subsequently, chimeric EGFR/Ufo receptors consisting of the extracellular domains of the epidermal growth factor receptor (EGFR) and the transmembrane and intracellular moiety of Ufo were engineered. Using different far-Western blot analyses and coimmunoprecipitation assays, receptor binding of PLCgamma and p85 proteins as well as GRB2, c-src and lck was examined in vitro and in vivo. Competitive inhibition of substrate binding and mutagenesis experiments with EGFR/Ufo constructs revealed C-terminal tyrosine 821 (EILpYVNMDEG) as a docking site for multiple effectors, namely PLCgamma, p85 proteins, GRB2, c-src and lck. Tyrosine 779 (DGLpYALMSRC) demonstrated an additional, but lower binding affinity for the p85 proteins in vitro. In addition, binding of PLCgamma occurred through tyrosine 866 (AGRpYVLCPST). Moreover, our in vivo data indicate that further direct or indirect binding sites for PLCgamma, GRB2, c-src and lck on the human Ufo receptor may exist.

Blotting, Western↗