Abstract
The H2a alternatively spliced variant of the human asialoglycoprotein receptor H2 subunit differs from the H2b variant by an extra pentapeptide, EGHRG, present in the ectodomain next to the membrane-span. This difference causes retention and degradation in the endoplasmic reticulum (ER) of H2a when expressed without the H1 subunit in 3T3 cells. In contrast, a significant portion of singly expressed H2b is Golgi-processed and reaches the cell surface. Using a new specific anti-H2a antibody, we found that in HepG2 cells, H2a is rapidly cleaved to a 35-kDa fragment, comprising the entire ectodomain, most of which is secreted into the medium. The cleavage site for the secreted fragment was located at the lumenal end of the membrane span. No membrane-bound H2a exits the ER, indicating that the pentapeptide is a signal for ER retention and degradation of the membrane form but does not hinder secretion of the cleaved soluble form. H2a does not form a membrane receptor complex with H1 as H2b does. H2a is therefore not a subunit of the receptor but a precursor for a secreted form of the protein; signal peptidase is probably responsible for the cleavage to the soluble fragment. Therefore, the juxtamembrane sequence regulates the function of the transmembrane domain of a type II membrane protein as either a signal-anchor sequence (H2b) or as a cleaved signal sequence, which generates a secreted product (H2a).
Highlights
The H2a alternatively spliced variant of the human asialoglycoprotein receptor H2 subunit differs from the H2b variant by an extra pentapeptide, EGHRG, present in the ectodomain next to the membrane-span
The asialoglycoprotein receptor (ASGPR) H2a subunit has been used as a model for studies on endoplasmic reticulum (ER) degradation, due to the fact that when the polypeptide was expressed in transfected 3T3 cells in the absence of the H1 subunit it was retained in the ER, in contrast to its alternatively spliced variant H2b [1, 2, 11]
We have studied the fate of H2a in HepG2 cells with the help of a new antibody that discriminates between H2a and H2b
Summary
Sandra Tolchinsky‡, Ming Huam Yuk§¶, Michal Ayalon‡, Harvey F. We have described the different subcellular fates of two alternatively spliced variants of the H2 subunit of the ASGPR, H2a and H2b, when expressed in NIH 3T3 cells without H1 These variants differ only by the presence in H2a of an extra 5-amino acid insert in the ectodomain adjacent to the membrane spanning region [1]. In the case of the ASGPR H2a, alternative splicing creates a transcript containing an extra mini-exon, which plays a role in the cleavage and secretion of the ectodomain as we describe here This result strengthens the theory that signal anchor domains on type II transmembrane proteins are functionally equivalent to signal peptides [19, 20]
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