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P Clapp

Publications and source records attributed to P Clapp.

7 recordsLinked to original sources
PubMed2001

Evidence that a protein-protein interaction 'hot spot' on heterotrimeric G protein betagamma subunits is used for recognition of a subclass of effectors.

To understand the requirements for binding to G protein betagamma subunits, phage-displayed random peptide libraries were screened using immobilized biotinylated betagamma as the target. Selected peptides were grouped into four different families based on their sequence characteristics. One group (group I) had a clear conserved motif that has significant homology to peptides derived from phospholipase C beta (PLC beta) and to a short motif in phosducin that binds to G protein beta subunits. The other groups had weaker sequence homologies or no homology to the group I sequences. A synthetic peptide from the strongest consensus group blocked activation of PLC by G protein betagamma subunits. The peptide did not block betagamma-mediated inhibition of voltage-gated calcium channels and had little effect on betagamma-mediated inhibition of Gs-stimulated type I adenylate cyclase. Competition experiments indicated that peptides from all four families bound to a single site on betagamma. These peptides may bind to a protein-protein interaction 'hot spot' on the surface of betagamma subunits that is used by a subclass of effectors.

Amino Acid Sequence↗
PubMed1999

HSP27 expression regulates CCK-induced changes of the actin cytoskeleton in CHO-CCK-A cells.

We investigated how heat shock protein 27 (HSP27) and its phosphorylation are involved in the action of cholecystokinin (CCK) on the actin cytoskeleton by genetic manipulation of Chinese hamster ovary (CHO) cells stably transfected with the CCK-A receptor. In these cells, as in rat acini, CCK activated p38 mitogen-activated protein (MAP) kinase and increased the phosphorylation of HSP27. This effect could be blocked with the p38 MAP kinase inhibitor SB-203580. Examination by confocal microscopy of cells stained with rhodamine phalloidin showed that CCK dose-dependently induced changes of the actin cytoskeleton, including cell shape changes, which were coincident with actin cytoskeleton fragmentation and formation of actin filament patches in the cells. To further evaluate the role of HSP27, CHO-CCK-A cells were transfected with expression vectors for either wild-type (wt) or mutant (3A, 3G, and 3D) human HSP27. Overexpression of wt-HSP27 and 3D-HSP27 inhibited the effects on the actin cytoskeleton seen after high-dose CCK stimulation. In contrast, overexpression of nonphosphorylatable mutants, 3A- and 3G-HSP27, or inhibition of phosphorylation of HSP27 by preincubation of wt-HSP27 transfected cells with SB-203580 did not protect the actin cytoskeleton. These results suggest that phosphorylation of HSP27 is required to stabilize the actin cytoskeleton and to protect the cells from the effects of high concentrations of CCK.

Actin Cytoskeleton↗
PubMed1998

Identification of the sites of N-linked glycosylation on the human calcium receptor and assessment of their role in cell surface expression and signal transduction.

The human calcium receptor (hCaR) is a G-protein-coupled receptor containing 11 potential N-linked glycosylation sites in the large extracellular domain. The number of potential N-linked glycosylation sites actually modified, and the effect on cell surface expression and signal transduction of blocking glycosylation at these sites, was examined by site-directed mutagenesis. Asparagine residues of the consensus sequences (Asn-Xaa-Ser/Thr) for N-linked glycosylation were mutated to glutamine individually and in various combinations to disrupt the potential N-linked glycosylation sites in the context of the full-length receptor. The cDNA constructs were transiently transfected into HEK-293 cells lacking endogeneous hCaR, and expressed receptors were analyzed by mobility differences on immunoblots, glycosidase digestion, intact cell enzyme-linked immunoassay, and extracellular calcium-stimulated phosphoinositide hydrolysis assay. Immunoblot analyses and glycosidase digestion studies of the wild type versus mutant receptors demonstrate that, of the 11 potential sites for N-linked glycosylation, eight sites (Asn-90, -130, -261, -287, -446, -468, -488, and -541) are glycosylated; the three remaining sites (Asn-386, -400, and -594) may not be efficiently glycosylated in the native receptor. Sequential mutagenesis of multiple N-linked glycosylation sites and analyses by immunoblotting, immunofluorescence, biotinylation of cell surface proteins, and intact cell enzyme-linked immunoassay indicated that disruption of as few as three glycosylation sites impairs proper processing and expression of the receptor at the cell surface. Disruption of five glycosylation sites reduced cell surface expression by 50-90% depending on which five sites were disrupted. Phosphoinositide hydrolysis assay results for various glycosylation-defective mutant receptors in general correlated well with the level of cell surface expression. Our results demonstrate that among 11 potential N-linked glycosylation sites on the hCaR, eight sites are actually utilized; glycosylation of at least three sites is critical for cell surface expression of the receptor, but glycosylation does not appear to be critical for signal transduction.

Amino Acid Substitution↗