Search

Although interactions between the mu2 subunit of the clathrin adaptor protein complex AP-2 and tyrosine-based internalization motifs have been implicated in the selective recruitment of cargo molecules into coated pits, the functional significance of this interaction for endocytosis of many types of membrane proteins remains unclear. To analyze the function of mu2-receptor interactions, we constructed an epitope-tagged mu2 that incorporates into AP-2 and is targeted to coated pits. Mutational analysis revealed that Asp176 and Trp421 of mu2 are involved in the interaction with internalization motifs of TGN38 and epidermal growth factor (EGF) receptor. Inducible overexpression of mutant mu2, in which these two residues were changed to alanines, resulted in metabolic replacement of endogenous mu2 in AP-2 complexes and complete abrogation of AP-2 interaction with the tyrosine-based internalization motifs. As a consequence, endocytosis of the transferrin receptor was severely impaired. In contrast, internalization of the EGF receptor was not affected. These results demonstrate the potential usefulness of the dominant-interfering approach for functional analysis of the adaptor protein family, and indicate that clathrin-mediated endocytosis may proceed in both a mu2-dependent and -independent manner.

In the developing central nervous system, LIM homeodomain codes control the development of cell types with Lhx3 involved in the production of cells for locomotion, motor neurons, and V2 interneurons. Data show that Lhx3 binds to LIM cofactor NLI triggering V2 interneuron differentiation. Furthermore, Lhx3 undergoes transformation from an interneuron-promoting factor to a motor neuron-promoting factor.

Heterotrimeric GTP- binding proteins (G proteins) control cellular functions by transducing signals from the outside to the inside of cells. Regulator of G protein signaling (RGS) proteins are key modulators of the amplitude and duration of G protein-mediated signaling through their ability to serve as guanosine triphosphatase-activating proteins (GAPs). We have identified RGS-PX1, a [Gα.sub.s]-specific GAP. The RGS domain of RGS-PX1 specifically interacted with [Gα.sub.s], accelerated its GTP hydrolysis, and attenuated [Gα.sub.s]-mediated signaling. RGS-PX1 also contains a Phox (PX) domain that resembles those in sorting nexin (SNX) proteins. Expression of RGS-PX1 delayed lysosomal degradation of the EGF receptor. Because of its bifunctional role as both a GAP and a SNX, RGS-PX1 may link heterotrimeric G protein signaling and vesicular trafficking., Heterotrimeric G proteins relay extracellular signals initiated by hormones, neurotransmitters, chemokines, and sensory stimuli through G protein-coupled receptors to intracellular effectors and trigger a variety of physiological responses (1, 2). [...]

Enzyme assays following cotransfection of membrane lipid desaturase (MLD) gene with epidermal growth factor receptor were utilized to identify the gene encoding a desaturase and to determine the effect on biosynthesis of the transmembrane receptor for epidermal growth factor (EGF). Results revealed that MLD has properties of a membrane protein of endoplasmic reticulum. It was also shown that MLD specifically inhibits the biosynthesis of the EGF receptor following overexpression in transfected cells.

The retroviral transfer method helps examine the biological effects of a mutant epidermal growth factor receptor (EGFR) lacking exons 2-7 (de 2-7 EGFR). This transfer helps the expression of de 2-7 EGFR in glioblastoma cells expressing wild-type endogenous receptors. Western blotting analysis reveals that these cells express endogenous 170-kDa EGFR and exogenous 140-155 kDa de 2-7 EGFR. Implementation of mutant EGFR in mice increases tumorigenic capacity. Tumor-specific alteration of EGFR influences interactions between tumor cells and their microenvironment.