CALEB/NGC Interacts with the Golgi-associated Protein PIST

119

150

We have previously identified the cystic fibrosis transmembrane regulator (CFTR)-interacting protein CAL and demonstrated that CAL modulates CFTR plasma membrane expression by retaining CFTR within the cell. Here, we report that in addition to regulating membrane expression, CAL also regulates the expression of mature CFTR. The co-expression of hemagglutinintagged or Myc-tagged CAL with green fluorescent protein (GFP)-CFTR in COS-7 cells causes a dose-dependent reduction in mature GFP-CFTR, independent of its tags. Bafilomycin A1, a lysosomal proton pump inhibitor, increases mature GFP-CFTR, confirming previous reports of lysosomal degradation of mature CFTR. Importantly, bafilomycin A1 reverses CAL-mediated CFTR degradation. The proteasome inhibitor, MG132, on the other hand, does not reverse the effect of CAL. CAL has no effect on CFTR maturation, suggesting that it exerts its effects on mature CFTR. Co-expression of CAL enhances the degradation of CFTR. We showed previously that CAL reduces the half-life of CFTR at the cell surface. Here we show that expression of dominant-negative dynamin 2 K44A, a large GTPase inhibitor that is known to inhibit clathrin-mediated endocytosis and vesicle formation in the Golgi, increases cell surface CFTR as measured by surface biotinylation. More importantly, dynamin 2 K44A also restores cell surface CFTR in CALoverexpressing cells and partially blocks the CAL-mediated degradation of mature CFTR. These data suggest a model in which CAL retains CFTR in the cell and targets CFTR for degradation.

supporting

confidence: 48%

Modulation of Mature Cystic Fibrosis Transmembrane Regulator Protein by the PDZ Domain Protein CAL

Cheng

Wang

Guggino

2004

119

150

“…All of these proteins play a role in the intracellular transport or targeting of their partner proteins (16,53,(57)(58)(59)(60)(61). The MALS family may be of particular relevance in providing insight into the role of AIPP1.…”

Section: Discussionmentioning

confidence: 99%

A Single PDZ Domain Protein Interacts with the Menkes Copper ATPase, ATP7A

Stephenson

Dubach

Lim

et al. 2005

The homeostatic regulation of essential elements such as copper requires many proteins whose activities are often mediated and tightly coordinated through protein-protein interactions. This regulation ensures that cells receive enough copper without intracellular concentrations reaching toxic levels. To date, only a small number of proteins implicated in copper homeostasis have been identified, and little is known of the protein-protein interactions required for this process. To identify other proteins important for copper homeostasis, while also elucidating the protein-protein interactions that are integral to the process, we have utilized a known copper protein, the copper ATPase ATP7A, as a bait in a yeast two-hybrid screen of a human cDNA library to search for interacting partners. One of the ATP7A-interacting proteins identified is a novel protein with a single PDZ domain. This protein was recently identified to interact with the plasma membrane calcium ATPase b-splice variants. We propose a change in name for this protein from PISP (plasma membrane calcium ATPase-interacting single-PDZ protein) to AIPP1 (ATPase-interacting PDZ protein) and suggest that it represents the protein that interacts with the class I PDZ binding motif identified at the ATP7A C terminus. The interaction in mammalian cells was confirmed and an additional splice variant of AIPP1 was identified. This study represents an essential step forward in identifying the proteins and elucidating the network of protein-protein interactions involved in maintaining copper homeostasis and validates the use of the yeast two-hybrid approach for this purpose.

“…In this respect, PIST might be a general sorting molecule for a wide variety of transmembrane proteins ranging from GPCRs (SSTR5 and the ␤ 1 -adrenergic receptor; Ref. 43) to cell adhesion molecules (44) to ion channels (chloride channel 3b) or their associated proteins such as stargazin (45). PDZK1 also recognizes multiple types of membrane proteins.…”

Section: Discussionmentioning

confidence: 99%

Interactions with PDZ Domain Proteins PIST/GOPC and PDZK1 Regulate Intracellular Sorting of the Somatostatin Receptor Subtype 5

Wente

Stroh

Beaudet

et al. 2005

By yeast two-hybrid screening we have identified interaction partners for the intracellular C-terminal tail of the human and rodent somatostatin receptor subtype 5 (SSTR5). Interactions with the PDZ domain-containing proteins PIST and PDZK1 are mediated by the PDZ ligand motif at the C terminus of the receptor; in case of the human and mouse (but not the rat) receptors, a slight sequence variation of this motif also allows for binding of the peroxisomal receptor PEX5. PIST is Golgi-associated and retains SSTR5 in the Golgi apparatus when coexpressed with the receptor; PDZK1 on the other hand associates with the SSTR5 at the plasma membrane. Endogenous SSTR5 in the neuroendocrine AtT-20 tumor cell line is colocalized with PIST in the Golgi apparatus. On a functional level, removal of the PDZ ligand motif of the receptor does not interfere with agonist-dependent internalization of the receptor or its targeting to a Golgi-associated compartment; however, recycling of the receptor to the plasma membrane after washout of the agonist is inhibited, suggesting that the PDZ-mediated interaction of SSTR5 is required for postendocytic sorting.In recent years it has been appreciated that the signaling and the subcellular distribution of G-protein-coupled receptors may be influenced by proteins that interact with the intracellular regions of the receptors, most notably the C termini (1, 2). Several interactions have been mapped to the membrane proximal domain of the C terminus, including the so-called helix 8, which encompasses the palmitoylation motifs found in most receptors of the rhodopsin-related type I family (3-5). These contacts especially involve proteins that act in the postendocytic, intracellular sorting of multiple receptors, i.e. the sorting nexin SNX1, N-ethylmaleimide sensitive factor and the G-protein-coupled receptor-associated sorting protein GASP. A second type of interactions involves the distal C termini of GPCRs, 4 which in many cases contain motifs for the interaction with PDZ domains. PDZ domain proteins frequently act as scaffold molecules because of their multiple protein interaction motifs (6). Thus this type of interaction has the potential to target a receptor to specific subcellular domains and into specific signaling complexes. Interaction of the ␤ 1 -adrenergic receptor with the third PDZ domain of PSD-95, for example, is believed to anchor this receptor at the postsynaptic density of excitatory synapses (7). The interaction of the parathyroid hormone receptor with Na ϩ /H ϩ exchanger regulatory factor/EBP-50 on the other hand physically links the receptor to phospholipase C␤, thereby shifting the second messenger response from adenylate cyclase activation to the hydrolysis of phospholipids (8).We have recently initiated a search for intracellular interaction partners for the various members of the somatostatin receptor (SSTR) family (9, 10). There are five SSTR subtypes in mammalian species, which form a rather homogeneous subfamily within the larger family of GPCRs, as they all preferential...