Hu-K4 is a ubiquitously expressed type 2 transmembrane protein associated with the endoplasmic reticulum.

Hu-K4 is a human protein homologous to the K4L protein of vaccinia virus. Due to the presence of two HKD motifs, Hu-K4 was assigned to the family of Phospholipase D proteins although so far no catalytic activity has been shown. The Hu-K4 mRNA is found in many human organs with highest expression levels in the central nervous system. We extended the ORF of Hu-K4 to the 5' direction. As a consequence the protein is 53 amino acids larger than originally predicted, now harbouring a putative transmembrane domain. The exon/intron structure of the Hu-K4 gene reveals extensive alternative splicing in the 5' untranslated region. Due to the absence of G/C-rich regions and upstream ATG codons, the mRNA isoform in brain may be translated with higher efficacy leading to a high Hu-K4 protein concentration in this tissue. Using a specific antiserum produced against Hu-K4 we found that Hu-K4 is a membrane-bound protein colocalizing with protein disulfide isomerase, a marker of the endoplasmic reticulum. Glycosylation of Hu-K4 as shown by treatment with peptide N-glycosidase F or tunicamycin indicates that Hu-K4 has a type 2 transmembrane topology.

SorLA signaling by regulated intramembrane proteolysis.

The single-transmembrane receptor SorLA/LR11 contains binding domains typical for lipoprotein receptors and a VPS10 domain, which binds the neuropeptide head-activator. This undecapeptide enhances proliferation of neuronal precursor cells in a SorLA-dependent manner. Using specific inhibitors we found previously that head activator activates shedding of SorLA by the metalloprotease TACE close to the transmembrane domain releasing the large extra-cellular part of the receptor. Here we show that the remaining COOH-terminal membrane fragment of SorLA is processed by gamma-secretase. Inhibition of gamma-secretase by specific inhibitors or overexpression of dominant negative presenilin mutants and knock out of the presenilin genes led to accumulation of the SorLA membrane fragment and also of full-length SorLA in the membrane. In an in vitro assay we observed the gamma-secretase-dependent release of the two soluble cleavage products, the SorLA cytoplasmic domain and the SorLA beta-peptide. These processing steps are reminiscent of a novel signaling pathway that has been described for the notch receptor. Here, the notch cytoplasmic domain is released into the cytoplasm by the gamma-secretase and migrates to the nucleus where it acts as a transcriptional regulator. In parallel we found that a fusion protein of the released cytoplasmic tail of SorLA with EGFP located to the nucleus only if the nuclear localization signal of SorLA was intact. In a reporter gene assay the cytoplasmic domain of SorLA acted as a transcriptional activator indicating that SorLA might directly regulate transcription after activation by gamma-secretase.