Generation of specific antibodies against the rap1A, rap1B and rap2 small GTP‐binding proteins

Abstract

Specific antibodies against rapl A and rapl B small GTP-binding proteins were generated by immunization of rabbits with peptides derived from the C-terminus of the processed proteins. Immunoblot analysis of membranes from several mammalian cell lines and human thrombocytes with affinity-purified antibodies against raplA or raplB demonstrated the presence of multiple immunoreactive proteins in the 22 -23 kDa range, although at strongly varying levels. Whereas both proteins were present in substantial amounts in membranes from myelocytic HL-60, K-562 and HEL cells, they were hardly detectable in membranes from lymphoma U-937 and S49.1 cyc-cells. Membranes from human thrombocytes and 3T3-Swiss Albino fibroblasts showed strong rapl B immunoreactivity, whereas raplA protein was present in much lower amounts. In the cytosol of HL-60 cells, only small amounts of raplA and raplB proteins were detected, unless the cells were treated with lovastatin, an inhibitor of hydroxymethylglutaryl-coenzyme A reductase, suggesting that both proteins are isoprenylated. By comparison with recombinant proteins, the ratio of rapl A/ras proteins in membranes from HL-60 cells was estimated to be about 4: 1. An antiserum directed against the Cterminus of rap2 reacted strongly with recombinant rap2, but not with membranes from tested mammalian cells. In conclusion, rapl A and rapl B proteins are distributed differentially among membranes from various mammalian cell types and are isoprenylated in HL-60 cells.The rap proteins belong to the rapidly growing family of small GTP-binding proteins [I -31. Up till now, amino acid sequences for four types of rap proteins have been deduced from cloned cDNA, namely raplA [4-71, raplB [8], rap2 [4] and rap2B [9]. Except for their C-terminal sequences, the rap proteins are highly similar to each other and share the effector domain with ras proteins [l -31.The C-terminus of ras proteins is essential for attaching the proteins to the plasma membrane, largely by modifications to the last four amino acids, the so-called CAAX motif. These modifications include attachment of a C15 (farnesyl) isoprenoid to the cysteine of the CAAX motif, removal of the final three amino acids and methylation of the newly exposed Ca group [lo]. More recently, it was shown that raplA protein expressed in insect cells [ll] and raplB protein purified from human platelets [12] are modified by a C20 (geranylgeranyl) isoprenoid.Little is known about the function of rap proteins in mammalian cells. Interest has focussed on the raplA (Krev-I) protein, whose cDNA was cloned first using the feature of its homology to ras proteins [4] and more recently by its ability

B Cell Antigen Receptor Signaling Induces the Formation of Complexes Containing the Crk Adapter Proteins

“…Subcellular Localization of Crk, Cas, and Cbl-Both Crk and C3G are cytosolic proteins, whereas Rap is targeted to the cytosolic face of cellular membranes by a lipid modification (35,36). The ability of Crk⅐C3G complexes to regulate Rap may therefore require translocation of these complexes from the cytoplasm to cellular membranes.…”

Rap1b is required for normal platelet function and hemostasis in mice

“…Stimulation of the Gα q -linked PAR4 activates phospholipase Cβ (PLCβ), leading to phosphoinositol hydrolysis and an intracellular calcium increase, providing a necessary and sufficient stimulus for Rap1b activation (13), which is later sustained by activated protein kinase C (15). ADP stimulates Rap1b activation via a calcium-independent pathway downstream from its Gα i -coupled P2Y 12 receptor and is dependent on activation of phosphoinositide 3-kinase (16,17) and its lipid product phosphatidylinositol 3,4,5-triphosphate (18).…”

“…While Rap1 proteins are ubiquitously expressed in tissues, Rap1b is the predominant Rap1 isoform and the most abundant Ras family member in platelets (12), where it becomes rapidly activated upon stimulation with a variety of agonists acting through distinct signaling pathways (13,14). Stimulation of the Gα q -linked PAR4 activates phospholipase Cβ (PLCβ), leading to phosphoinositol hydrolysis and an intracellular calcium increase, providing a necessary and sufficient stimulus for Rap1b activation (13), which is later sustained by activated protein kinase C (15).…”

Purinergic A2b Receptor Activation by Extracellular Cues Affects Positioning of the Centrosome and Nucleus and Causes Reduced Cell Migration

“…Rap1B is both cytosolic and membrane-associated (67). Its distribution within cells, like Epac1, varies.…”