Identification of the endophilins (SH3p4/p8/p13) as novel binding partners for the β1-adrenergic receptor

Tang, Yuting; Hu, Liaoyuan A.; Miller, William E.; Ringstad, Niels; Hall, Randy A.; Pitcher, Julie A.; DeCamilli, Pietro; Lefkowitz, Robert J.

doi:10.1073/pnas.96.22.12559

Cited by 135 publications

(106 citation statements)

References 35 publications

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“…Other structures that could recruit endophilin include caveolae (34) or micropinocytic vesicles. Although dynamin was recruited to CCPs independent of endophilin expression, the accumulation of SJ1-145 to CCPs strongly depended on the expression of endophilin, consistent with an important role of endophilin in the targeting of SJ1 (20,21,23 tial association by its BAR domain, with the curved membrane of the vesicle stalk (5,18,25,26); (ii) interaction by its SH3 domain with dynamin (15); and (iii) binding, again by its SH3 domain, with cargo proteins present in the nascent vesicle (35)(36)(37). Because endophilin exists as a dimer (38), it may simultaneously bind two different proteins, e.g., synaptojanin and either vesicle cargo or dynamin, by the two SH3 domains of the dimer.…”

Section: Discussionmentioning

confidence: 89%

Two synaptojanin 1 isoforms are recruited to clathrin-coated pits at different stages

Perera¹,

Zoncu

Lucast

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

155

187

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Phosphoinositides are thought to play an important role in clathrincoated pit (CCP) dynamics. Biochemical and structural studies have shown a direct interaction of phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P 2] with endocytic clathrin adaptors, whereas functional studies using cell-free systems or intact cells have demonstrated the importance of PI(4,5)P 2 synthesis and dephosphorylation in clathrin coating and uncoating, respectively. Furthermore, genetic manipulations of kinases and phosphatases involved in PI(4,5)P 2 metabolism result in major defects in synaptic vesicle recycling and other forms of clathrin-dependent endocytosis. However, live imaging studies of these enzymes at CCPs have not been conducted. We have used multicolor total internal reflection fluorescence microscopy (TIRFM) to visualize the spatialtemporal recruitment of synaptojanin 1 (SJ1), a polyphosphoinositide phosphatase, and its binding partner endophilin to CCPs. Strikingly, we observed differential temporal recruitment of the two major SJ1 splice variants to CCPs. The 145-kDa isoform, the predominant isoform expressed in the brain, was rapidly recruited as a ''burst,'' together with endophilin, at a late stage of CCP formation. In contrast, the nonneuronal ubiquitously expressed 170-kDa isoform of SJ1 was present at all stages of CCP formation. These results raise the possibility that dynamic phosphoinositide metabolism may occur throughout the lifetime of a CCP.dynamin ͉ endocytosis ͉ endophilin ͉ phosphoinositides ͉ phosphatidylinositol (4,5)-bisphosphate

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Section: Discussionmentioning

confidence: 89%

Two synaptojanin 1 isoforms are recruited to clathrin-coated pits at different stages

Perera¹,

Zoncu

Lucast

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

155

187

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“…Also surprising to us was the results of overlay assays suggesting that in HEK 293 cells and in brain Alix is the main endophilin interactor. Indeed, endophilins are known partners of metalloprotease disintegrins MDC5 and MDC 9 and of the ␤ 1 -adrenergic receptor, which should be absent from our extracts since they are transmembrane proteins (20,21). They also bind to the 145-kDa synaptojanin 1 and to two proteins migrating around 100 kDa, dynamin and germinal center kinase-like kinase (12,14,19), and to amphiphysins I and II, migrating around 120 and 80 kDa.…”

Section: Discussionmentioning

confidence: 99%

Alix (ALG-2-interacting Protein X), a Protein Involved in Apoptosis, Binds to Endophilins and Induces Cytoplasmic Vacuolization

Chatellard-Causse¹,

Blot²,

Cristina³

et al. 2002

Journal of Biological Chemistry

159

142

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ALG-2-interacting protein X (Alix), also known as AIP1, is a cytoplasmic protein ubiquitously expressed and concentrated in phagosomes and exosomes. Alix may regulate apoptosis since it binds apoptosis-linked gene 2 (ALG-2), a Ca 2؉ -binding protein necessary for cell death, and also overexpression of its C-terminal half (Alix-CT) blocks death induced by several stimuli. This part of Alix contains a long proline-rich domain containing several potential SH3-binding sites. Using Alix as bait in a yeast two-hybrid system to screen a mouse brain library, we have found that SH3p4, SH3p8, and SH3p13, collectively known as endophilins, bind to Alix. Co-immunoprecipitations and overlay experiments allowed us to demonstrate that endophilins bind to Alix-CT through an SH3/proline-rich domain interaction. We have narrowed the region of Alix interacting with endophilins down to 14 amino acids containing a PXRPPPP consensus sequence, also present in synaptojanin and germinal center kinase-like kinase, allowing their interaction to endophilins. We further show that overexpression of Alix-CT, which blocks cell death, leads to cytoplasmic vacuolization into tubulo-vesicular structures delineated by Alix-CT. This vacuolization phenomenon is greatly enhanced upon co-expression with endophilins and may be part of the protecting mechanism afforded by Alix-CT.

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“…One candidate is the proline-rich region in the third intracellular loop of ␤ 1 AR that is recognized by Src homology 3 domain-containing proteins of the endophilin family (53). Overexpression of endophilin (53) or deletion of the region (31) enhances ␤ 1 AR internalization. Another candidate is the N-terminal region that contains an allelic polymorphism at codon 49.…”

Section: Discussionmentioning

confidence: 99%

Resistance of the Human β1-Adrenergic Receptor to Agonist-mediated Down-regulation

Liang

Austin²,

Hoang

et al. 2003

Journal of Biological Chemistry

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Prolonged agonist stimulation results in down-regulation of most G protein-coupled receptors. When we exposed baby hamster kidney cells stably expressing the human ␤ 1 -adrenergic receptor (␤ 1 AR) to agonist over a 24-h period, we instead observed an increase of ϳ30% in both ␤ 1 AR binding activity and immune-detected receptors. In contrast, ␤ 2 AR expressed in these cells exhibited a decrease of >50%. We determined that the basal turnover rates of the two subtypes were similar (t1 ⁄2 ϳ 7 h) and that agonist stimulation increased ␤ 2 AR but not ␤ 1 AR turnover. Blocking receptor trafficking to lysosomes with bafilomycin A 1 had no effect on basal turnover of either subtype but blocked agonist-stimulated ␤ 2 AR turnover. As ␤ 1 AR mRNA levels increased in agonist-stimulated cells, ␤ 1 AR up-regulation appeared to result from increased synthesis with no change in degradation. To explore the basis for the subtype differences, we expressed chimeras in which the C termini had been exchanged. Each chimera responded to persistent agonist stimulation based on the source of its C-tail; ␤ 1 AR with a ␤ 2 AR C-tail underwent down-regulation, and ␤ 2 AR with a ␤ 1 AR C-tail underwent up-regulation. The C-tails had a corresponding effect on agonist-stimulated receptor phosphorylation and internalization with the order being ␤ 2 AR > ␤ 1 AR with ␤ 2 AR C-tail > ␤ 2 AR with a ␤ 1 AR C-tail > ␤ 1 AR. As internalization may be a prerequisite for down-regulation, we addressed this possibility by co-expressing each subtype with arrestin-2. Although ␤ 1 AR internalization was increased to that of ␤ 2 AR, down-regulation still did not occur. Instead, ␤ 1 AR accumulated inside the cells. We conclude that in unstimulated cells, both subtypes appear to be turned over by the same mechanism. Upon agonist stimulation, both subtypes are internalized, and ␤ 2 AR but not ␤ 1 AR undergoes lysosomal degradation, the fate of each subtype being regulated by determinants in its C-tail.

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Identification of the endophilins (SH3p4/p8/p13) as novel binding partners for the β1-adrenergic receptor

Cited by 135 publications

References 35 publications

Two synaptojanin 1 isoforms are recruited to clathrin-coated pits at different stages

Two synaptojanin 1 isoforms are recruited to clathrin-coated pits at different stages

Alix (ALG-2-interacting Protein X), a Protein Involved in Apoptosis, Binds to Endophilins and Induces Cytoplasmic Vacuolization

Resistance of the Human β1-Adrenergic Receptor to Agonist-mediated Down-regulation

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