Mediated Membrane-Binding Proteins

Calcium-Mediated Membrane-Binding Proteins A. Introduction 

Calcium-mediated membrane binding of proteins occurs both inside cells and outside. At first one might think that these events should be very different in these two different environments, with calcium concentrations in (jlM and mM, respectively. It is, however, not necessarily so. 

Functionally, recoverin acts by binding to rhodopsin kinase and inhibits rhodopsin phosphorylation (88). It has also been observed to bind rod outer segment (ROS) membranes (89). The interaction with rhodopsin kinase does not require amino-terminal acylation, but binding to ROS membranes is acylation dependent, suggesting two distinct binding sites and/or binding modes on recoverin. 

The annexins form a group of mainly intracellular proteins with a Ca2 -dependent binding to phospholipids. No clear physiological role of the annexins has so far been defined, but a wide range of biological functions have been suggested (90-97). Amino acid sequence analysis 

A schematic diagram of the conformational change and extrusion of the myri-stoyl group from the N-terminal domain of recoverin upon calcium binding followed by insertion into the membrane. 

None bound to only neutral phospholipid vesicles. The difference between IV and the other was localized to an Asp-to-Ser substitution and verified by mutant studies. Falke and co-workers (137) have shown that an isolated C2 domain from CPIA2 binds Ca and binds to phosphatidylcholine vesicles in a Ca -dependent manner. The domain binds two Ca in the absence as well as in the presence of phospholipid vesicles however, the binding affinity increases from Kd = 24 p,M in the absence of vesicles to = 3 p,M in the presence of vesicles. 

Stereo view of C2 jaws of the apo and Sm forms of PLC-(51. The apo form is shown with dark backbone and light side chains, and the Sm form with light backbone and dark side chains. (Redrawn with permission from Grobler et al. 1996.) 

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III. Calcium-Mediated Membrane-Binding Proteins A. Introduction... 

Annexins Phospholipid- and membrane-binding proteins involved in the regulation of cell growth, coagulation, mediation of secretion, signal transduction, and ion channel activity link signaling to membrane dynamics... 

Fig. 1. The GP Ib-IX-V complex. The complex consists of seven transmembrane polypeptides denoted GP Iba (mol wt 145,000), GP IbP (mol wt 24,000), GPIX (mol wt 17,000) and GP V (mol wt 82,000), in a stoichiometry of 2 2 2 1. The hatched region represents the plasma membrane. The area above the hatched region represents the extracellular space that below represents the cytoplasm. The complex is a major attachment site between the plasma membrane and the cytoskeleton. Two molecules associated with the cytoplasmic domain are depicted a 14-3-3 dimer, which may mediate intracellular signaling, and actin-binding protein, which connects the complex to the cortical cytoskeleton and fixes its position and influences its function.

C2 domains (phosphokinase C conserved 2 domains) mediate membrane targeting of diverse peripheral proteins. A C2 domain consists of approximately 130 residues and was first discovered as the Ca2+-binding site in conventional phosphokinase Cs. 

Conventional and novel PKC isozymes are potently activated by phorbol esters, heterocyclic compounds found in the milky sap exuded by plants of the Euphorbiaccae family. This sap was used medicinally as a counterirritant and cathartic agent over the millennia we now know that the active ingredients, phorbol esters, specifically bind to the Cl domain, the diacylglycerol sensor described above. In fact, their ability to recruit PKC to membranes is so effective that phorbol esters cause maximal activation of conventional PKCs, bypassing the requirement for Ca2+. This module is found in a number of other proteins in addition to PKC, so the profound effects of phorbol esters on cells are mediated by other proteins in addition to PKC. 

SNAPs is an acronym for soluble NSF attachment proteins. They were originally discovered as cofactors for NSF that mediate the membrane binding of NSF in in vitro transport assays. Several isoforms of SNAPs exist in mammalian cells. SNAPs are also highly conserved proteins. Crystallographic studies indicated that the proteins form a very stiff and twisted sheet that is formed by a series of antiparallel and tightly packed helices connected by short loops. 

PTKs can be subdivided into two large families, receptor tyrosine kinases (RTKs) and non-RTKs. The human genome encodes for a total of 90 tyrosine kinases of which 32 are nonreceptor PTKs that can be placed in 10 subfamilies (Fig. 1). All nonreceptor PTKs share a common kinase domain and usually contain several additional domains that mediate interactions with protein-binding partners, membrane lipids, or DNA (Table 1). These interactions may affect cellular localization and the activation status of the kinase or attract substrate proteins for phosphorylation reactions. 

Figure 1. Simplified schematic of receptor-mediated signal transduction in neutrophils. Binding of ligand to the receptor activates a guanine-nucleotide-binding protein (G protein), which then stimulates phospholipase C. Phosphatidylinositol 4,5-bis-phosphate is cleaved to produce diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). DAG stimulates protein kinase C. IP3 causes the release of Ca from intracellular stores, which results in an increase in the cytosolic Ca concentration. This increase in Ca may stimulate protein kinase C, calmodulin-dependent protein kinases, and phospholipase A2. Protein phosphorylation events are thought to be important in stimulating degranulation and oxidant production. In addition, ionic fluxes occur across the plasma membrane. It is possible that phospholipase A2 and ionic channels may be governed by G protein interactions. ...

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