Macropinocytosis: Discovery of Macropinocytosis Gene Agpa and Therapeutic Potential

Macropinocytosis: Discovery of macropinocytosis gene AgpA and therapeutic potential

Macropinocytosis is a process by which cells engulf large volumes of fluid. It allows for non-selective uptake of macromolecules, and in some cases bacteria and viruses; this differs from particle and receptor-driven uptake such as phagocytosis . Macropinocytosis generally accompanies cell ruffling, and can be mediated by several pathways including actin-driven protrusions. For some cell types it is a constitutive process, and is inducible in cell types and populations that do not normally macropinocytose.

In experiments attempting to identify new genes implicated in macropinocytosis, Charito et al. used restriction enzymes to integrate a plasmid genome into Dictyostelium discoideum in attempt to induce mutants that do not properly macropinocytose. The surviving Dictyostelium discoideum were grown in bacterial media and screened for defects in fluid uptake; 50% of the surviving mutants were found to have defects in fluid uptake. The mutants were shown to uptake red fluorescent dextran slowly compared to the wild-type; this indicated a severe deficiency in terms of the mutants ability to macropinocytose within a red fluorescent dextran media. Inverse PCR was then used to extract pieces of the genes mutated from the restriction-enzyme mediated integration. The fragments were then isolated, purified and sequenced; the sequenced gene was compared to previously sequenced genes within a genetic database in attempt to identify it, and potential homologs.

The gene was idenfitied as AgpA, and encoded a glycerol phosphate acyltransferase, which converts lysophosphatidic acid to phosphatidic acid. AgpA null mutants, which have increased lysophosphatidic acid levels and decreased phosphatidic acid levels, have several altered behaviors because of the changes in concentration of the two acids including decreased macropinocytosis. Lower phosphatidic acid levels also result in less phosphatidyl inositol (PI); PI is the precursor to phosphatidyl inositol triphosphate (PIP3) which in turn activates Protein Kinase B (PKB), a protein implicated in activation of macropinocytosis.

In wild-type Dictyostelium, cell speed remains relatively constant, whereas AgpA null mutants speed increases to nearly eight times that of wild-type cells over similar time periods. This results from increased levels of lysophosphatidic acid, which increases Rho GTPase activation . This increased GTPase activation ultimately leads to higher levels of actin polymerization that causes amplified filopodia and lamellipodia formation; the end result being a higher cell velocity and more cell motility. In addition, AgpA null mutants do not experience normal growth in vitro, they grow slowly, compared to wild-type cells that exhibit a natural logarithmic growth rate. The AgpA null mutants also have a tendency to shrink in size over time.