Calnexin, an endoplasmic reticulum transmembrane protein, represents a new type of molecular chaperone that selectively associates in a transient fashion with newly synthesized monomeric glycoproteins in HepG2 cells. Calnexin only recognizes glycoproteins when they are incompletely folded. Dissociation of glycoproteins from calnexin occurs at different rates and is related to the time taken for their folding, which may then initiate their differential transport rates from the endoplasmic reticulum.

Rat liver parenchyma harbors equal numbers of epidermal growth factor (EGF) and insulin receptors. Following administration of a saturating dose of EGF (10 micrograms/100 g body weight), there was a rapid (t1/2 approximately 1.1 min) internalization of receptor coincident with its tyrosine phosphorylation at residue 1173 and receptor recruitment of the adaptor protein SHC, its tyrosine phosphorylation and its association with GRB2 and the Ras guanine nucleotide exchange factor, mSOS, largely in endosomes. This led to a cytosolic pool of a complex of tyrosine‐phosphorylated SHC, GRB2 and mSOS. It was demonstrated that these constituents were linked to Ras activation by the characteristic decrease in Raf‐1 mobility on SDS‐PAGE, which was maintained for 60 min after a single bolus of administered EGF. While insulin administration (15 micrograms/100 g body weight) led to insulin receptor beta‐subunit tyrosine phosphorylation and internalization, there was little detectable tyrosine phosphorylation of SHC, recruitment of GRB2, association of a complex with mSOS or any detectable change in the mobility of Raf‐1. Therefore, in normal physiological target cells in vivo, distinct signaling pathways are realized after EGF or insulin receptor activation, with regulation of this specificity most probably occurring at the locus of the endosome.

A shuttle system has been developed to genetically encode unnatural amino acids in mammalian cells using aminoacyl-tRNA synthetases (aaRSs) evolved in E. coli. A pyrrolysyl-tRNA synthetase (PylRS) mutant was evolved in E. coli that selectively aminoacylates a cognate nonsense suppressor tRNA with a photocaged lysine derivative. Transfer of this orthogonal tRNA-aaRS pair into mammalian cells made possible the selective incorporation of this unnatural amino acid into proteins.

The type I membrane protein calnexin functions as a molecular chaperone for secretory glycoproteins in the endoplasmic reticulum with ATP and Ca2+ as two of the cofactors involved in substrate binding. Protease protection experiments with intact canine rough microsomes showed that amino acid residues 1-462 of calnexin are located within the lumen of the endoplasmic reticulum. Expression using the baculovirus Sf9 insect cell system of a recombinant truncated calnexin corresponding to residues 1-462 (calnexin delta TMC) revealed an association in vivo with a coexpressed secretory glycoprotein substrate, human immunodeficiency virus type I gp120. For the in vitro characterization of calnexin delta TMC, we purified this secreted form to homogeneity from the medium of Sf9 cells. We demonstrate that the properties of the purified calnexin delta TMC correspond to those of full-length calnexin in canine microsomes with at least one intramolecular disulfide bond and binding to 45Ca2+. Calnexin delta TMC underwent a marked and reversible conformational change following Ca2+ binding as measured by its resistance to proteinase K digestion of a 60-kDa fragment and also by the change from an oligomeric form of calnexin delta TMC to a monomeric form. We also found that calnexin bound Mg-ATP leading to a conformational change from a monomeric to an oligomeric form that coincided as with markedly increased proteinase sensitivity. Our results identify the luminal domain of calnexin as responsible for binding substrates, Ca2+, and Mg-ATP. Because Ca2+ and ATP are required in vivo for the maintenance of calnexin-substrate interactions, conformational changes in the luminal domain of calnexin induced by Ca2+ and Mg-ATP are relevant to the in vivo function of calnexin as a molecular chaperone.