Biomedical subjects

Junqi He

Publications and source records attributed to Junqi He.

6 recordsLinked to original sources

PubMed2005

Proteomic analysis of beta1-adrenergic receptor interactions with PDZ scaffold proteins.

Many G protein-coupled receptors possess carboxyl-terminal motifs ideal for interaction with PDZ scaffold proteins, which can control receptor trafficking and signaling in a cell-specific manner. To gain a panoramic view of beta1-adrenergic receptor (beta AR) interactions with PDZ scaffolds, the beta1AR carboxyl terminus was screened against a newly developed proteomic array of PDZ domains. These screens confirmed beta1AR associations with several previously identified PDZ partners, such as PSD-95, MAGI-2, GIPC, and CAL. Moreover, two novel beta1AR-interacting proteins, SAP97 and MAGI-3, were also identified. The beta1AR carboxyl terminus was found to bind specifically to the first PDZ domain of MAGI-3, with the last four amino acids (E-S-K-V) of beta1AR being the key determinants of the interaction. Full-length beta1AR robustly associated with full-length MAGI-3 in cells, and this association was abolished by mutation of the beta1AR terminal valine residue to alanine (V477A), as determined by co-immunoprecipitation experiments and immunofluorescence co-localization studies. MAGI-3 co-expression with beta1AR profoundly impaired beta1AR-mediated ERK1/2 activation but had no apparent effect on beta1AR-mediated cyclic AMP generation or agonist-promoted beta1AR internalization. These findings revealed that the interaction of MAGI-3 with beta1AR can selectively regulate specific aspects of receptor signaling. Moreover, the screens of the PDZ domain proteomic array provide a comprehensive view of beta1AR interactions with PDZ scaffolds, thereby shedding light on the molecular mechanisms by which beta1 AR signaling and trafficking can be regulated in a cell-specific manner.

Amino Acid Sequence↗

PubMed2004

Interaction with cystic fibrosis transmembrane conductance regulator-associated ligand (CAL) inhibits beta1-adrenergic receptor surface expression.

G protein-coupled receptors such as the beta1-adrenergic receptor (beta1AR) must be trafficked to the plasma membrane in order to bind with their extracellular ligands and regulate cellular physiology. By using glutathione S-transferase pull-down techniques, we found that the beta1AR carboxyl terminus directly interacts with the cystic fibrosis transmembrane conductance regulator-associated ligand (CAL; also known as PIST, GOPC, and FIG), a protein known to be primarily localized to the Golgi apparatus. CAL contains two predicted coiled-coil domains and one PSD-95/Discs-large/ZO-1 homology (PDZ) domain. The beta1AR carboxyl terminus (CT) binds to the PDZ domain of CAL, with the last few amino acids (ESKV) of the beta1AR-CT being the key determinants for the interaction. Mutation of the terminal valine residue resulted in markedly reduced association of the beta1AR-CT with CAL. Numerous other mutations to the ESKV motif also impaired the beta1AR-CT/CAL interaction, suggesting that this motif is close to optimal for association with the CAL PDZ domain. In cells, full-length beta1AR robustly associates with CAL, and this interaction is abolished by mutation of the terminal valine to alanine of the receptor (V477A), as determined by co-immunoprecipitation experiments and immunofluorescence co-localization studies. Consistent with observations that CAL is a Golgi-associated protein, overexpression of CAL reduces surface expression of beta1AR. Interaction with CAL promotes retention of beta1AR within the cell, whereas PSD-95, another beta1AR-associated PDZ domain-containing protein, competitively blocks beta1AR association with CAL and promotes receptor trafficking to the cell surface. These data reveal that CAL, a novel beta1AR-binding partner, modulates beta1AR intracellular trafficking, thereby revealing a new mechanism of regulation for beta1AR anterograde trafficking through the endoplasmic reticulum-Golgi complex to the plasma membrane.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Cell cycle-dependent phosphorylation of Disabled-2 by cdc2.

Disabled-2 (Dab2; also known as p96 and DOC-2) is a signal transduction protein that has been implicated in the control of cell growth. Dab2 is known to be a phosphoprotein, but little is known about the kinases that phosphorylate Dab2. We have found that Dab2 phosphorylation is markedly increased during the mitosis phase of the cell cycle. This phosphorylation is blocked by roscovitine, a selective inhibitor of cyclin-dependent kinases. Dab2 robustly coimmunoprecipitates from cells with the cyclin-dependent kinase cdc2, and purified cdc2 can phosphorylate purified Dab2 fusion proteins in vitro on multiple sites. Cellular phosphorylation of Dab2 by cdc2 promotes the association of Dab2 with Pin1, a peptidylprolyl isomerase that regulates the rate of Dab2 dephosphorylation. These findings reveal that Dab2 is differentially phosphorylated during the cell cycle by cdc2 and provide a potential feedback mechanism by which Dab2 inhibition of cell growth and proliferation may be regulated.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Heterodimerization of alpha 2A- and beta 1-adrenergic receptors.

beta- and alpha(2)-adrenergic receptors are known to exhibit substantial cross-talk and mutual regulation in tissues where they are expressed together. We have found that the beta(1)-adrenergic receptor (beta(1)AR) and alpha(2A)-adrenergic receptor (alpha(2A)AR) heterodimerize when coexpressed in cells. Immunoprecipitation studies with differentially tagged beta(1)AR and alpha(2A)AR expressed in HEK-293 cells revealed robust co-immunoprecipitation of the two receptors. Moreover, agonist stimulation of alpha(2A)AR was found to induce substantial internalization of coexpressed beta(1)AR, providing further evidence for a physical association between the two receptors in a cellular environment. Ligand binding assays examining displacement of [(3)H]dihydroalprenolol binding to the beta(1)AR by various ligands revealed that beta(1)AR pharmacological properties were significantly altered when the receptor was coexpressed with alpha(2A)AR. Finally, beta(1)AR/alpha(2A)AR heterodimerization was found to be markedly enhanced by a beta(1)AR point mutation (N15A) that blocks N-linked glycosylation of the beta(1)AR as well as by point mutations (N10A/N14A) that block N-linked glycosylation of the alpha(2A)AR. These data reveal an interaction between beta(1)AR and alpha(2A)AR that is regulated by glycosylation and that may play a key role in cross-talk and mutual regulation between these receptors.

Cells, Cultured↗

PubMed2002

Disabled-2 is essential for endodermal cell positioning and structure formation during mouse embryogenesis.

The signal transduction adapter protein Disabled-2 (Dab2) is one of the two mammalian orthologs of the Drosophila Disabled. The brain-specific Disabled-1 (Dab1) functions in positional organization of brain cells during development. Dab2 is widely distributed and is highly expressed in many epithelial cell types. The dab2 gene was interrupted by in-frame insertion of beta-galactosidase (LacZ) in embryonic stem cells and transgenic mice were produced. Dab2 expression was first observed in the primitive endoderm at E4.5, immediately following implantation. The homozygous Dab2-deficient mutant is embryonic lethal (earlier than E6.5) due to defective cell positioning and structure formation of the visceral endoderm. In E5.5 dab2 (-/-) conceptus, visceral endoderm-like cells are present in the deformed primitive egg cylinder; however, the visceral endoderm cells are not organized, the cells of the epiblast have not expanded, and the proamniotic cavity fails to form. Disorganization of the visceral endodermal layer is evident, as cells with positive visceral endoderm markers are scattered throughout the dab2 (-/-) conceptus. Only degenerated remains were observed at E6.5 for dab2 (-/-) embryos, and by E7.5, the defective embryos were completely reabsorbed. In blastocyst in vitro culture, initially cells with characteristics of endoderm, trophectoderm, and inner cell mass were observed in the outgrowth of the hatched dab2 (-/-) blastocysts. However, the dab2 (-/-) endodermal cells are much more dispersed and disorganized than those from wild-type blastocysts, the inner cell mass fails to expand, and the outgrowth degenerates by day 7. Thus, Dab2 is required for visceral endodermal cell organization during early mouse development. The absence of an organized visceral endoderm in Dab2-deficient conceptus leads to the growth failure of the inner cell mass. We suggest that Dab2 functions in a signal pathway to regulate endodermal cell organization using endocytosis of ligands from the blastocoel cavity as a positioning cue.

Adaptor Proteins, Signal Transducing↗

PubMed2002

Glycosylation of beta(1)-adrenergic receptors regulates receptor surface expression and dimerization.

The beta(1)-adrenergic receptor (beta(1)AR) has one predicted site of N-linked glycosylation on its extracellular amino-terminus, but the glycosylation and potential functional importance of this site have not yet been examined. We show here that the beta(1)AR is glycosylated in various cell types and that mutation of the single predicted site of N-linked glycosylation (N15A) results in the formation of receptors that are not N-glycosylated. The beta(1)AR N15A mutant exhibited significantly decreased basal surface expression relative to the wild-type receptor but had no detectable deficits in ligand binding or agonist-promoted internalization. Co-immunoprecipitation experiments using Flag-tagged and HA-tagged receptors demonstrated that the beta(1)AR-N15A mutant receptor exhibits a markedly reduced capacity for dimerization relative to wild-type beta(1)AR. These data reveal that the beta(1)AR is glycosylated on Asn15 and that this glycosylation plays a role in regulating beta(1)AR surface expression and dimerization.

Amino Acid Substitution↗