David H. Llewellyn

Nuclear localisation of calreticulin in vivo is enhanced by its interaction with glucocorticoid receptors

© 1997 Federation of European Biochemical Societies.

Homozygous acute intermittent porphyria: compound heterozygosity for adjacent base transitions in the same codon of the porphobilinogen deaminase gene

SummaryA sister and brother with severe porphobilinogen (PBG) deaminase deficiency are described. Each of their parents carries a different mutation for acute intermittent porphyria and the children are homozygous for the PBG-deaminase deficiency that causes this disorder. Both are compound heterozygotes for adjacent base transitions in the same codon in exon 10 of the PBG deaminase gene.

Role of calreticulin in regulating intracellular Ca2+ storage and capacitative Ca2+ entry in HeLa cells

Calreticulin is a Ca2+ binding protein located primarily in the endoplasmic reticulum (ER) lumen of non-excitable cells, where it is considered to be involved mainly in Ca2+ storage and buffering. However, there is increasing evidence to implicate the protein in other facets of Ca2+ signalling. In this study, we sought to establish more clearly the role of the protein in the regulation of intracellular Ca2+ signalling. Generating HeLa cells stably transfected with GFP-tagged calreticulin (GFPCRT) allowed to us to select cells by FACS in which calreticulin was expressed at ten times its endogenous levels. Using transiently expressed aequorin as a Ca2+ indicator in these cells, we investigated the role of calreticulin in intracellular Ca2+ storage, IP3-mediated Ca2+ release, and capacitative Ca2+ entry. The data showed that the capacity of the ionomycin-sensitive Ca2+ store was doubled in over-expressing cells, indicating that although calreticulin has a role in Ca2+ storage within the lumen, other lumenal proteins are also likely to be involved. No difference was observed in the release of Ca2+ from the IP3-sensitive store in response to prolonged single stimulation with histamine in the absence of extracellular Ca2+, but use of short, sequential pulses of histamine and ATP revealed that calreticulin may exert an effect upon IP3-mediated Ca2+ release. Two different experimental approaches indicated that calreticulin participates in the regulation of capacitative Ca2+ entry. In the presence of extracellular Ca2+, the histamine-generated cytosolic Ca2+ signal was significantly lower in GFPCRT cells than those in control cells. Induction of capacitative Ca2+ entry by complete emptying of the store using the SERCA pump inhibitor, cyclopiazonic acid also showed that the influx component was significantly reduced in the GFPCRT cells. Use of ER-targeted apoaequorin acting as a luciferase demonstrated that the resting ER free [Ca2+] in the GFPCRT cells was lower than that in control cells. These data implicate calreticulin in the control of IP3-mediated Ca2+ release and capacitative Ca2+ entry, which may involve direct interaction with Ca2+ signalling components or control of ER free [Ca2+].

Acute intermittent porphyria caused by a C→T mutation that produces a stop codon in the porphobilinogen deaminase gene

SummaryA mutation of the porphobilinogen (PBG) deaminase gene that produces the cross-reacting immunological material (CRIM)-negative type of acute intermittent porphyria (AIP) has been identified in one of 43 unrelated patients with this form of the disorder. The mutation is a C→T transition that abolishes a PstI recognition site in exon 9 of the gene and converts a codon for glutamine to a stop codon.

Calreticulin comes of age

The workshop organizers thank the following organizations for financial support, making possible the provision of bursaries to PhD students and junior post-doctoral workers: the Wellcome Trust, Amersham Pharmacia Biotech, Promega UK, Novus and abcam. We also thank the conference secretariat, Alison Marsland, who made an invaluable contribution in helping to make the workshop a success.