Biomedical subjects

Urszula Golebiewska

Publications and source records attributed to Urszula Golebiewska.

5 recordsLinked to original sources

PubMed2007

Membrane-permeable calmodulin inhibitors (e.g. W-7/W-13) bind to membranes, changing the electrostatic surface potential: dual effect of W-13 on epidermal growth factor receptor activation.

Membrane-permeable calmodulin inhibitors, such as the napthalenesulfonamide derivatives W-7/W-13, trifluoperazine, and calmidazolium, are used widely to investigate the role of calcium/calmodulin (Ca2+/CaM) in living cells. If two chemically different inhibitors (e.g. W-7 and trifluoperazine) produce similar effects, investigators often assume the effects are due to CaM inhibition. Zeta potential measurements, however, show that these amphipathic weak bases bind to phospholipid vesicles at the same concentrations as they inhibit Ca2+/CaM; this suggests that they also bind to the inner leaflet of the plasma membrane, reducing its negative electrostatic surface potential. This change will cause electrostatically bound clusters of basic residues on peripheral (e.g. Src and K-Ras4B) and integral (e.g. epidermal growth factor receptor (EGFR)) proteins to translocate from the membrane to the cytoplasm. We measured inhibitor-mediated translocation of a simple basic peptide corresponding to the calmodulin-binding juxtamembrane region of the EGFR on model membranes; W-7/W-13 causes translocation of this peptide from membrane to solution, suggesting that caution must be exercised when interpreting the results obtained with these inhibitors in living cells. We present evidence that they exert dual effects on autophosphorylation of EGFR; W-13 inhibits epidermal growth factor-dependent EGFR autophosphorylation under different experimental conditions, but in the absence of epidermal growth factor, W-13 stimulates autophosphorylation of the receptor in four different cell types. Our interpretation is that the former effect is due to W-13 inhibition of Ca2+/CaM, but the latter results could be due to binding of W-13 to the plasma membrane.

Animals↗

PubMed2006

Structure of the membrane reconstituted transmembrane-juxtamembrane peptide EGFR(622-660) and its interaction with Ca2+/calmodulin.

The transmembrane (TM) and juxtamembrane (JM) regions of the epidermal growth factor receptor (EGFR) couple ligand binding in the extracellular domain to activation of the kinase domain. Solid-state NMR and polarized FTIR measurements of peptides corresponding to the TM plus JM regions of EGFR (residues 622-660) reconstituted in model phospholipid membranes are presented to address the role of the short cytoplasmic JM sequence (residues 645-660) in regulating EGFR activity. We show that the TM domain is helical with a transition to non-helical structure at the TM-JM boundary. Fluorescence measurements indicate that the JM region of EGFR(622-660) binds to the membrane surface and that binding can be reversed by the addition of the complex of Ca2+ and calmodulin. Together these data support models suggesting the cytoplasmic JM region of EGFR plays an active role in regulating receptor activity.

Amino Acid Sequence↗

PubMed2006

Membrane-bound basic peptides sequester multivalent (PIP2), but not monovalent (PS), acidic lipids.

Several biologically important peripheral (e.g., myristoylated alanine-rich C kinase substrate) and integral (e.g., the epidermal growth factor receptor) membrane proteins contain clusters of basic residues that interact with acidic lipids in the plasma membrane. Previous measurements demonstrate that the polyvalent acidic lipid phosphatidylinositol 4,5-bisphosphate is bound electrostatically (i.e., sequestered) by membrane-adsorbed basic peptides corresponding to these clusters. We report here three experimental observations that suggest monovalent acidic lipids are not sequestered by membrane-bound basic peptides. 1), Binding of basic peptides to vesicles does not decrease when the temperature is lowered below the fluid-to-gel phase transition. 2), The binding energy of Lys-13 to lipid vesicles increases linearly with the fraction of monovalent acidic lipids. 3), Binding of basic peptides to vesicles produces no self-quenching of fluorescent monovalent acidic lipids. One potential explanation for these results is that membrane-bound basic peptides diffuse too rapidly for the monovalent lipids to be sequestered. Indeed, our fluorescence correlation spectroscopy measurements show basic peptides bound to phosphatidylcholine/phosphatidylserine membranes have a diffusion coefficient approximately twofold higher than that of lipids, and those bound to phosphatidylcholine/phosphatidylinositol 4,5-bisphosphate membranes have a diffusion coefficient comparable to that of lipids.

Amino Acid Sequence↗

PubMed2005

Reversible - through calmodulin - electrostatic interactions between basic residues on proteins and acidic lipids in the plasma membrane.

The inner leaflet of a typical mammalian plasma membrane contains 20-30% univalent PS (phosphatidylserine) and 1% multivalent PtdIns(4,5)P(2). Numerous proteins have clusters of basic (or basic/hydrophobic) residues that bind to these acidic lipids. The intracellular effector CaM (calmodulin) can reverse this binding on a wide variety of proteins, including MARCKS (myristoylated alanine-rich C kinase substrate), GAP43 (growth-associated protein 43, also known as neuromodulin), gravin, GRK5 (G-protein-coupled receptor kinase 5), the NMDA (N-methyl-D-aspartate) receptor and the ErbB family. We used the first principles of physics, incorporating atomic models and the Poisson-Boltzmann equation, to describe how the basic effector domain of MARCKS binds electrostatically to acidic lipids on the plasma membrane. The theoretical calculations show the basic cluster produces a local positive electrostatic potential that should laterally sequester PtdIns(4,5)P(2), even when univalent acidic lipids are present at a physiologically relevant 100-fold excess; four independent experimental measurements confirm this prediction. Ca(2+)/CaM binds with high affinity (K(d) approximately 10nM) to this domain and releases the PtdIns(4,5)P(2). MARCKS, a major PKC (protein kinase C) substrate, is present at concentrations comparable with those of PtdIns(4,5)P(2) (approx. 10 microM) in many cell types. Thus MARCKS can act as a reversible PtdIns(4,5)P(2) buffer, binding PtdIns(4,5)P(2) in a quiescent cell, and releasing it locally when the intracellular Ca(2+) concentration increases. This reversible sequestration is important because PtdIns(4,5)P(2) plays many roles in cell biology. Less is known about the role of CaM-mediated reversible membrane binding of basic/hydrophobic clusters for the other proteins.

Animals↗

PubMed2002

Lateral sequestration of phosphatidylinositol 4,5-bisphosphate by the basic effector domain of myristoylated alanine-rich C kinase substrate is due to nonspecific electrostatic interactions.

A peptide corresponding to the basic (+13), unstructured effector domain of myristoylated alanine-rich C kinase substrate (MARCKS) binds strongly to membranes containing phosphatidylinositol 4,5-bisphosphate (PIP(2)). Although aromatic residues contribute to the binding, three experiments suggest the binding is driven mainly by nonspecific local electrostatic interactions. First, peptides with 13 basic residues, Lys-13 and Arg-13, bind to PIP(2)-containing vesicles with the same high affinity as the effector domain peptide. Second, removing basic residues from the effector domain peptide reduces the binding energy by an amount that correlates with the number of charges removed. Third, peptides corresponding to a basic region in GAP43 and MARCKS effector domain-like regions in other proteins (e.g. MacMARCKS, adducin, Drosophila A kinase anchor protein 200, and N-methyl-d-aspartate receptor) also bind with an energy that correlates with the number of basic residues. Kinetic measurements suggest the effector domain binds to several PIP(2). Theoretical calculations show the effector domain produces a local positive potential, even when bound to a bilayer with 33% monovalent acidic lipids, and should thus sequester PIP(2) laterally. This electrostatic sequestration was observed experimentally using a phospholipase C assay. Our results are consistent with the hypothesis that MARCKS could reversibly sequester much of the PIP(2) in the plasma membrane.

Amino Acid Sequence↗