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Biomedical subjects

K A Lund

Publications and source records attributed to K A Lund.

7 recordsLinked to original sources
PubMed1998

Differential signaling and regulation of apical vs. basolateral EGFR in polarized epithelial cells.

Overexpression of the epidermal growth factor receptors (EGFR) in polarized kidney epithelial cells caused them to appear in high numbers at both the basolateral and apical cell surfaces. We utilized these cells to look for differences in the regulation and signaling of apical vs. basolateral EGFR. Apical and basolateral EGFR were biologically active and mediated EGF-induced cell proliferation to similar degrees. Receptor downregulation and endocytosis were less efficient at the apical surface, resulting in prolonged EGF-induced tyrosine kinase activity at the apical cell membrane. Tyrosine phosphorylation of EGFR substrates known to mediate cell proliferation, Src-homologous and collagen protein (SHC), extracellularly regulated kinase 1 (ERK1), and ERK2 could be induced similarly by activation of apical or basolateral EGFR. Focal adhesion kinase was tyrosine phosphorylated more by basolateral than by apical EGFR; however, beta-catenin was tyrosine phosphorylated to a much greater degree following the activation of mislocalized apical EGFR. Thus EGFR regulation and EGFR-mediated phosphorylation of certain substrates differ at the apical and basolateral cell membrane domains. This suggests that EGFR mislocalization could result in abnormal signal transduction and aberrant cell behavior.

Animals↗
PubMed1990

Phosphorylation of the epidermal growth factor receptor at threonine 654 inhibits ligand-induced internalization and down-regulation.

To assess the functional significance of phosphorylation of the epidermal growth factor (EGF) receptor at Thr654, we compared the effects of 12-O-tetradecanoyl-13-acetate (TPA) on ligand-induced internalization and down-regulation between wild-type and mutant receptors that contain an alanine substitution at position 654. Activation of protein kinase C with TPA blocked EGF-induced internalization and down-regulation of Thr654 receptors and inhibited in vivo tyrosine kinase activity by 80%. TPA did not inhibit transferrin receptor internalization or constitutive EGF receptor internalization, suggesting that protein kinase C activation inhibits only the ligand-induced process. Inhibition by TPA of induced internalization, down-regulation, and kinase activity required threonine at position 654 since full-length Ala654 EGF receptors were significantly resistant to TPA inhibition of these ligand-induced activities. However, C'-terminal truncation further enhanced this resistance to TPA inhibition. The EGF-dependent internalization of kinase-inactive receptors truncated at residue 1022 was also impaired by TPA in Thr654 receptors, but not in Ala654 receptors, indicating that phosphorylation at Thr654 also interferes with tyrosine kinase-independent receptor activities. We conclude that the dominant regulatory effect of protein kinase C on the EGF receptor is mediated through phosphorylation at Thr654 which effectively inactivates the receptor. The submembrane region of the EGF receptor appears to regulate transmission of conformational information from the extracellular ligand-binding site to the cytoplasmic kinase and regulatory domains.

Alanine↗
PubMed1990

Quantitative analysis of the endocytic system involved in hormone-induced receptor internalization.

We have developed a quantitative method to evaluate the interaction between cell surface receptors and the endocytic apparatus. This method exploits occupancy-dependent changes in internalization rates that occur in cells expressing high numbers of receptors. We found that constitutive internalization of the transferrin receptor behaves as a simple, first order process that is unaltered by ligand. Internalization of the epidermal growth factor (EGF) receptor, however, behaves as a saturable, second order process that is induced by receptor occupancy. Internalization of EGF receptors occurs through at least two distinct pathways: a low capacity pathway that has a relatively high affinity for occupied receptors, and a low affinity pathway that has a much higher capacity. The high affinity pathway was observed in all cells having receptors with intrinsic tyrosine kinase activity. Mutant EGF receptors lacking kinase activity could not utilize the high affinity pathway and were internalized only through the low affinity one. Mutated receptors with decreased affinity for kinase substrates were also internalized at decreased rates through the high affinity, inducible pathway. In the case of vitellogenin receptors in Xenopus oocytes, occupied receptors competed more efficiently for internalization than empty ones. Insulin increased the endocytic capacity of oocytes for vitellogenin receptors. Similarly, serum increased the capacity of the inducible pathway for EGF receptors in mammalian cells. These data are consistent with a model of internalization in which occupied receptors bind to specific cellular components that mediate rapid internalization. Ligand-induced internalization results from an increase in the affinity of occupied receptors for the endocytic apparatus. Hormones can also indirectly regulate endocytosis by increasing the number of coated pits or their rate of internalization. The ability to dissect receptor-specific effects from cell-specific ones should be very useful in investigating the molecular mechanisms of receptor mediated endocytosis.

Animals↗
PubMed1989

Global modulation of the epidermal growth factor receptor is triggered by occupancy of only a few receptors. Evidence for a binary regulatory system in normal human fibroblasts.

We investigated the effect of epidermal growth factor (EGF) pretreatment on binding to its own receptor. We found that EGF specifically induces a rapid, reversible, and global change in the affinity of surface EGF receptors. Occupancy of only a few (less than 1%) was sufficient to reduce the affinity of the majority of surface receptors by 10 min and a maximal response required only 5% occupancy. The rate at which EGF receptor affinity decreased was essentially independent of the extent of receptor occupancy and occurred with a t 1/2 between 2-2.5 min. Surface receptors remained in the lower affinity state as long as EGF remained present. Removal of EGF resulted in the restoration of receptor affinity with a t 1/2 of about 20 min. Kinetic analyses revealed that the alteration in apparent affinity was due to changes in both the association and dissociation rate constants as well as an increase in the specific internalization rate of the receptor. Treatment of cells with phorbol esters produced a similar affinity drop, but depletion of intracellular protein kinase C did not affect the affinity change induced by EGF. Thus, phorbol esters and EGF mediate their effects through different pathways. EGF reduced the affinity of its own receptors in a variety of cell types including Chinese hamster ovary cells expressing transfected human EGF receptors. Our observations are consistent with the hypothesis that occupancy of a few receptors on EGF naive cells triggers a global modification/phosphorylation of surface receptors which results in the observed change in affinity. This system is independent of protein kinase C and probably serves to regulate the activity of the EGF receptor.

Animals↗
PubMed1989

Functional independence of the epidermal growth factor receptor from a domain required for ligand-induced internalization and calcium regulation.

We have located the distal boundary of the tyrosine kinase domain of the EGF receptor and have identified a distinct sequence in the C' terminus required for EGF-dependent receptor internalization, leading to receptor down-regulation and degradation. Within this receptor domain, an 18 amino acid highly negatively charged region of predicted helical structure is required both for endocytosis via a high-affinity, saturable pathway and for ligand-stimulated increases in cytosolic calcium. In contrast to kinase-inactive, internalization-competent receptors, kinase-active, internalization-defective receptors effectively signaled gene transcription, morphological transformation, and growth. These observations support the hypothesis that mitogenic responses to EGF are mediated by activation of the intrinsic protein tyrosine kinase activity of the membrane-bound receptor, with ligand-induced internalization serving to terminate the signal.

Amino Acid Sequence↗