ABSTRACT
Gamma-protocadherins (Pcdh gamma) are type I transmembrane proteins, which are most notably expressed in the nervous system. They are enriched at synapses and involved in synapse formation, specification, and maintenance. In this study, we show that Pcdh gamma C3 and Pcdh gamma B4 are specifically cleaved within their ectodomains by the disintegrin and metalloprotease ADAM10. Analysis of ADAM10-deficient fibroblasts and embryos, inhibitor studies, as well as RNA interference-mediated down-regulation demonstrated that ADAM10 is not only responsible for the constitutive but also for the regulated shedding of these proteins in fibroblasts and in neuronal cells. In contrast to N-cadherin shedding, which was activated by N-methyl-D-aspartic acid receptor activation in neuronal cells, Pcdh gamma shedding was induced by alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid hydrate stimulation, suggesting differential regulation mechanisms of cadherin-mediated functions at synapses. Cell aggregation assays in the presence or absence of metalloprotease inhibitors strongly suggest that the ectodomain shedding events modulate the cell adhesion role of Pcdh gamma. The identification of ADAM10 as the protease responsible for constitutive and regulated Pcdh gamma shedding may therefore provide new insight into the regulation of Pcdh gamma functions.
Subject(s)
ADAM Proteins/physiology , Cadherins/metabolism , Cell Adhesion , Membrane Proteins/physiology , ADAM Proteins/metabolism , ADAM10 Protein , Amyloid Precursor Protein Secretases , Animals , Blotting, Western , Cadherin Related Proteins , Cell Line , Cells, Cultured , Fibroblasts/cytology , Glutamic Acid/pharmacology , Humans , K562 Cells , Membrane Proteins/metabolism , Mice , Mice, Knockout , Neurons/cytology , SynapsesABSTRACT
Cadherins have been known for a long time to be key elements in many important biological processes. In particular, the role of classical cadherins in mediating adhesion has been examined in great detail. Over recent years, the accumulation of experimental tools and mice mutants has allowed more refined analysis of cadherin functions, and new aspects such as signaling and synapse dynamics have become the center of interest. In addition, the study of mice lacking the entire protocadherin-gamma cluster shed the first light on a possible novel function of members of this cadherin family in synapse formation and cell survival during development.
Subject(s)
Cadherins/physiology , Cell Death/physiology , Synapses/physiology , Animals , Cadherins/metabolism , Mammals , Mice , Models, Biological , Morphogenesis/physiology , Signal Transduction/physiologyABSTRACT
The recently described protocadherin gene clusters encode cadherin-related proteins, which are highly expressed in the vertebrate nervous system. Here, we report biochemical studies addressing proteolytic processing of gamma-protocadherins. These type-I transmembrane proteins are cleaved by a metalloproteinase in vivo, generating a soluble extracellular fragment and a carboxyl-terminal fragment associated with the cellular membrane. In addition, we show that the carboxyl-terminal fragment is a substrate for further cleavage mediated by presenilin. Consequently, accumulation of the fragment is found when gamma-secretase is inactivated either by the specific presenilin-inhibitor L685,458 or in double mutant murine embryonic fibroblasts lacking both presenilin genes. The gamma-secretase-generated carboxyl-terminal fragment is largely unstable but accumulates when proteasomal degradation is inhibited. Interestingly, the proteolytic fragment generated by gamma-secretase can localize to the nucleus. This is the first report providing experimental evidence for a cell surface receptor signaling function of protocadherins regulated by proteolytic events.
Subject(s)
Cadherins/metabolism , Membrane Proteins/metabolism , Amyloid Precursor Protein Secretases , Animals , Aspartic Acid Endopeptidases , COS Cells , Cadherin Related Proteins , Carbamates/pharmacology , Cell Line , Chlorocebus aethiops , Dipeptides/pharmacology , Endopeptidases/metabolism , Fibroblasts , Humans , Kidney , Matrix Metalloproteinases/metabolism , Mice , Presenilin-1 , Presenilin-2 , Protease Inhibitors/pharmacology , Protein Processing, Post-Translational , Recombinant Proteins/metabolism , TransfectionABSTRACT
The lumenal endoplasmic reticulum (ER) protein BiP, among its other functions, is believed to serve as an ER stress sensor, triggering the so-called 'unfolded protein response' or UPR. For this role, BiP levels are critical. Indeed, here we show that BiP expression is tightly controlled at a post-transcriptional level. Thus, an artificial increase in cellular BiP mRNA does not lead to increased synthesis of BiP in unstressed cells, and, consequently, protein levels remain constant. Under ER stress conditions, however, this homeostatic restriction is alleviated, and independent of transcript levels, the translation efficiency of BiP transcripts is enhanced, allowing the cells to produce more protein. We additionally show that this regulation is independent of elements in the 5' and 3' UTR of BiP mRNA, which rather points to a novel type of translational feedback control. BiP is the first example of a lumenal protein whose expression is controlled at a translational level. The implications of these findings with respect to cellular stress are discussed.
