Helix II and a conserved HPD tripeptide located in the J-domain bind to Hsp70 at an acidic groove located in the ATPase domain to stimulate ATP hydrolysis ( Greene et al., 1998 Suh et al., 1998). Hsp40s specify the jobs of Hsp70 by modulating Hsp70s ATP hydrolytic cycle, by acting as chaperone proteins that target substrates to Hsp70 and by influencing Hsp70s subcellular localization.Īll Hsp40 family members contain a J-domain that is ∼70 amino acids in length, is constructed from four α-helical regions, and is responsible for interactions with Hsp70 ( Hill et al., 1995 Qian et al., 1996). Hsp70 performs its cellular work by using the energy derived from ATP hydrolysis to bind and release protein substrates that display nonnative structure. Hsp40s direct Hsp70 to facilitate cellular processes that include protein folding, the suppression of amyloid plaque formation, endocytosis, protein translocation across membranes, signal transduction, DNA replication, protein degradation, and prion propagation ( Silver and Way, 1993 Cyr et al., 1994 Cheetham and Caplan, 1998 Hartl and Hayer-Hartl, 2002). Ydj1 and Sis1 contain exchangeable chaperone modules that assist in specification of Hsp70 function. In in vivo studies, YSY exhibited a gain of function and, unlike Ydj1, could complement the lethal phenotype of sis1Δ and facilitate maintenance of the prion. Purified SYS and YSY exhibited protein-folding activity and substrate specificity that mimicked that of Ydj1 and Sis1, respectively. To test whether the chaperone modules of Ydj1 and Sis1 function in the specification of Hsp70 action, we constructed a set of chimeric Hsp40s in which the chaperone domains of Ydj1 and Sis1 were swapped to form YSY and SYS. Ydj1 and Sis1 share a high degree of sequence identity in their amino and carboxyl terminal ends, but each contains a structurally unique and centrally located protein module that is implicated in chaperone function. The mechanism by which Ydj1 and Sis1 specify Hsp70 functions is not clear. Type I and type II Hsp40s, such as yeast Ydj1 and Sis1, form chaperone pairs with cytosolic Hsp70 Ssa1 that fold proteins with different efficiencies and carry out specific cellular functions. Our data suggest that optimal receptor-calnexin interactions critically regulate D 1 and D 2 receptor trafficking and expression at the cell surface, a mechanism likely to be of importance for many GPCRs.Hsp40 family members regulate Hsp70s ability to bind nonnative polypeptides and thereby play an essential role in cell physiology. Additionally, we show that calnexin interacts with the receptors via two distinct mechanisms, glycan-dependent and glycan-independent, which may underlie the multiple effects (ER retention and surface trafficking) of calnexin on receptor expression. This is likely because of an increase in ER retention because confocal microscopy revealed intracellular clustering of dopamine receptors that were co-localized with an ER marker protein. Overexpression of calnexin also results in a marked decrease in both D 1 and D 2 receptor expression. Confocal fluorescence microscopy reveals the accumulation of D 1-green fluorescent protein and D 2-yellow fluorescent protein receptors within internal stores following treatment with calnexin inhibitors. Inhibition of glycosylation either through receptor mutations or treatments with glycosylation inhibitors partially blocks the interactions with calnexin with a resulting decrease in cell surface receptor expression. To determine the influence of calnexin on receptor expression, we conducted assays in HEK293T cells using a variety of calnexin-modifying conditions. These protein-protein interactions were confirmed using Western blot analysis and co-immunoprecipitation experiments. We have now identified the ER chaperone protein calnexin as an interacting protein for both D 1 and D 2 dopamine receptors.
The mechanisms involved in the biogenesis and trafficking of GPCRs from the ER to the cell surface are poorly understood, but they may involve interactions with other proteins. As for all proteins, G protein-coupled receptors (GPCRs) undergo synthesis and maturation within the endoplasmic reticulum (ER).