GRP94 is a member of the
heat shock protein family normally confined to the endoplasmic reticulum that sometimes escapes the KDEL-mediated retention system. It is overexpressed in some gastric and other gastrointestinal
carcinomas, but little is known about the physiological role of
GRP94 in gastric mucosa. We investigated the membrane presence of
GRP94 in parietal cells, which secrete
acid into the gastric lumen, using subcellular fractionation, selective solubilization of
membrane proteins, Western blotting, and radio-
ligand binding and provided evidence of functional
GRP94 expression at the surface of gastric mucosa parietal cells anchored to the basolateral domain. Our results show that
GRP94 is not an
integral membrane protein since 50 mM Na2CO3 treatment dissociates part of it from the membrane. However, 100 mM Na2CO3 treatment did not extract all
GRP94 from the membrane, which indicates that it is strongly associated with it. The presence of
GRP94 in isolated plasma membrane was demonstrated by Western blotting and its functionality by radio-
ligand binding experiments. Both the K(D) value obtained in saturation experiments with N-ethylcarboxamido-[3H]
adenosine at 4°C, at the nanomolar range, and the inhibition constant of its binding by
radicicol, the most specific
GRP94 inhibitor, indicate that active receptor regions are exposed at the membrane surface. Western blotting of plasma membrane subfractions showed that
GRP94 is mainly expressed in the basolateral membrane of gastric parietal cells, while its presence in the apical domain is negligible, thereby inferring a role for
GRP94 in processes operating in this membrane domain.