GRP94: An HSP90-like protein specialized for protein folding and quality control in the endoplasmic reticulum

Abstract

Glucose-regulated protein 94 is the HSP90-like protein in the lumen of the endoplasmic reticulum and therefore it chaperones secreted and membrane proteins. It has essential functions in development and physiology of multicellular organisms, at least in part because of this unique clientele. GRP94 shares many biochemical features with other HSP90 proteins, in particular its domain structure and ATPase activity, but also displays distinct activities, such as calcium binding, necessitated by the conditions in the endoplasmic reticulum. GRP94's mode of action varies from the general HSP90 theme in the conformational changes induced by nucleotide binding, and in its interactions with co-chaperones, which are very different from known cytosolic co-chaperones. GRP94 is more selective than many of the ER chaperones and the basis for this selectivity remains obscure. Recent development of molecular tools and functional assays has expanded the spectrum of clients that rely on GRP94 activity, but it is still not clear how the chaperone binds them, or what aspect of folding it impacts. These mechanistic questions and the regulation of GRP94 activity by other proteins and by post-translational modification differences pose new questions and present future research avenues. This article is part of a Special Issue entitled: Heat Shock Protein 90 (HSP90).

Figure 1. Phylogenetic analysis of some HSP90 family members

The analysis is based on sequence comparisons of human cytosolic HSP90α (AAI21063.1) and HSP90β (NP_031381.2), human GRP94 (AAH66656.1), Arabidopsis thaliana GRP94 (NP_974606.1) and TRAP-1 (NP_057376.2), yeast HSP82 (P02829.1) and HSC82 (AAA02813.1), E. coli HtpG (AP_001122.1) and Leishmania infantum GRP94 (ADP89477.1) and Trypanosoma cruzi GRP94 by the algorithm MUSCLE, using the web site T-Coffee. The GRP94s are highlighted in red. Numbers at the nodes are bootstrapping values to validate the analysis, performed using PhyML3.0. The X axis is indicating the expected amount of sequence change, which is a combination of time and inherent mutation rate. Inherent mutation rate can be different for each genome, complicating the comparisons. Bar, 0.1 units.

Figure 2. Comparison of structural features between GRP94 and HSP90β

Schematic representation of the domain organization of human GRP94 and human HSP90β. Blue, N-terminal domain (NTD). Beige, charged linker domain. Orange, middle domain (MD). Brown, C-terminal domain (CTD). The similarity between them is indicated by the percent identity indicated for each of the 4 domains. Grey, the signal sequence of GRP94. Yellow and red, unique sequences that distinguish the two members of the family. KDEL, the C-terminal tetrapeptide of GRP94 that serves as the ER retention/retrival ligand for the KDEL receptor [20]. MEEVD, the C-terminal peptide of HSP90, which serves as the target for binding of various TPR containing proteins. ATP, the nucleotide binding site. CHO, the N-glycosylation site of GRP94. Ca⁺⁺, the known calcium binding activity of the charged linker of GRP94 [92].

Figure 3. Comparison of the promoter of GRP94 with other ER chaperone genes

A. Schematic representation of the promoter of human GRP94, compared to other ER chaperone genes. The ERSEs in each promoter are indicated by boxes and are numbered. Arrows indicate the orientation of the ERSEs whose function is orientation-independent. The numbers below the ERSEs define the bp position relative to the transcription start site (TSS). The dark grey boxes depict perfectly matched ERSEs, and the light grey boxes - imperfectly homologous ERSE, see below. GRP94 promoter has two canonical ERSEs, BiP and PDIA6 have one. In contrast, the PDIA1 promoter of a poorly inducible gene in mammals, has only two mismatched ERSEs. Note: the scheme is not to scale. B. Sequence comparison of the ERSEs of the indicated genes. The CCACG 3′ motif of ERSE-2 and -4 of the GRP94 promoter have one base mismatch with the consensus sequence. The PDIA1 ERSE-1 contains two mismatches.

Figure 4. Comparisons of the linker domains of human HSP90β (hum90β) and GRP94 (hum94)

*, identical residues. :, similar residues. Blue, acidic residues. Red, basic residues. Purple, Asn and Gln. Green, aromatic residues. Orange, aliphatic residues. Brown, Pro. Black, small side chain residues. Strands 8 and 9, which flank much of the charged stretches and bind the domain to the N-terminal domain are indicated, and the Trp zipper domain interaction motif within strand 9 is underlined. The heavily negative sequence in the middle of the GRP94 domain is likely to contain a Ca⁺⁺ binding site [92]. A human GRP94 allele has Pro³⁰⁰Leu substitution (yellow highlight) (http://browser.1000genomes.org/index.html, rs116891695). This sequence comparison is instructive because a 3D structure for the charged linkers has proven elusive.

Fig. 5. An IGF-dependent cellular assay for the function of GRP94 variants

Differential survival of grp94−/− cells in serum-free medium when transiently expressing GRP94-GFP chimeric constructs. WT GRP94-GFP confers the highest survival. ΔK is an in-frame deletion of amino acids 144–488 that is a chaperone-dead negative control. The point mutant D128N is incapable of binding ATP [170] and Q427A is partially deficient in ATP hydrolysis in vitro [86]. This figure is adapted from [108].