Differential regulation of macropinocytosis by Abi1/Hssh3bp1 isoforms.
BACKGROUND
Macropinocytosis, which is a constitutive cellular process of fluid and macromolecule uptake, is regulated by actin cytoskeleton rearrangements near the plasma membrane. Activation of Rac1, which is proposed to act upstream of the actin polymerization regulatory Wave 2 complex, has been found to correlate with enhanced macropinocytosis. One of the components of the Wave 2 complex is Abi1. Multiple, alternatively spliced isoforms of Abi1 are expressed in mammalian cells, but the functional significance of the various isoforms is unknown.


RESULTS
Here, using flow cytometric assay analysis for Alexa Fluor 647, we demonstrate that Abi1 isoforms 2 and 3 differentially regulate macropinocytosis. LNCaP cells expressing isoform 3 had increased macropinocytic uptake that correlated with enhanced cell spreading and higher Rac1 activation in comparison to cells expressing isoform 2. Isoform 2 expressing cells had decreased macropinocytic uptake, but demonstrated greater sensitivity to Rac1 activation. Moreover, more isoform 2 was localized within the cytoplasm in comparison to isoform 3, which was more associated with the plasma membrane. Activated Rac1 was found to specifically bind to a site in exon 10 of isoform 2 in vitro. Because of alternative mRNA splicing, exon 10 is absent from isoform 3, precluding similar binding of activated Rac1. Both isoforms, however, bound to inactive Rac1 through the same non-exon 10 site. Thus, Abi1 isoform 3-containing Wave 2 complex exhibited a differential binding to activated vs. inactive Rac1, whereas isoform 2-containing Wave 2 complex bound activated or inactive Rac1 comparably.


CONCLUSIONS
Based on these observations, we postulate that Abi1 isoforms differentially regulate macropinocytosis as a consequence of their different relative affinities for activated Rac1 in Wave 2 complex. These findings also raise the possibility that isoform-specific roles occur in other Abi1 functions.
Dubielecka
Patrycja M
PM
Laboratory of Cell Signaling, New York Blood Center, New York, New York, United States of America.
Cui
Ping
P
Xiong
Xiaoling
X
Hossain
Sajjad
S
Heck
Susanne
S
Angelov
Lyudmil
L
Kotula
Leszek
L
D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D002460:Cell Line; D002465:Cell Movement; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000378:metabolism; D003598:Cytoskeletal Proteins / Q000737:chemistry / Q000378:metabolism*; D004789:Enzyme Activation; D006801:Humans; D008954:Models, Biological; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D010641:Phenotype; D010873:Pinocytosis; D011485:Protein Binding; D025941:Protein Interaction Mapping; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism; D021381:Protein Transport; D011994:Recombinant Proteins / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal / Q000378:metabolism; D020830:rac1 GTP-Binding Protein / Q000378:metabolism
C513733:ABI1 protein, human; D048868:Adaptor Proteins, Signal Transducing; D003598:Cytoskeletal Proteins; D050505:Mutant Proteins; D020033:Protein Isoforms; D011994:Recombinant Proteins; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal; C067297:8-aminohexylamino cAMP; D000242:Cyclic AMP; D020830:rac1 GTP-Binding Protein