Abstract
BackgroundThe receptor for activated C-kinase 1 (RACK1) is a conserved protein belonging to the WD40 repeat family of proteins. It folds into a beta propeller with seven blades which allow interactions with many proteins. Thus it can serve as a scaffolding protein and have roles in several cellular processes.ResultsWe identified the product of the Dictyostelium discoideum gpbB gene as the Dictyostelium RACK1 homolog. The protein is mainly cytosolic but can also associate with cellular membranes. DdRACK1 binds to phosphoinositides (PIPs) in protein-lipid overlay and liposome-binding assays. The basis of this activity resides in a basic region located in the extended loop between blades 6 and 7 as revealed by mutational analysis. Similar to RACK1 proteins from other organisms DdRACK1 interacts with G protein subunits alpha, beta and gamma as shown by yeast two-hybrid, pulldown, and immunoprecipitation assays. Unlike the Saccharomyces cerevisiae and Cryptococcus neoformans RACK1 proteins it does not appear to take over Gβ function in D. discoideum as developmental and other defects were not rescued in Gβ null mutants overexpressing GFP-DdRACK1. Overexpression of GFP-tagged DdRACK1 and a mutant version (DdRACK1mut) which carried a charge-reversal mutation in the basic region in wild type cells led to changes during growth and development.ConclusionDdRACK1 interacts with heterotrimeric G proteins and can through these interactions impact on processes specifically regulated by these proteins.
Highlights
The receptor for activated C-kinase 1 (RACK1) is a conserved protein belonging to the WD40 repeat family of proteins
Blast results showed that GpbB is highly related to the RACK1 family of proteins and the alignment of RACK1 sequences from diverse organisms such as H. sapiens, D. melanogaster, A. thaliana, D. discoideum and S. cerevisiae revealed significant sequence identity
The greatest difference is observed between propeller blades 6 and 7 where an extended loop of mainly basic amino acids is present in the D. discoideum and the A. thaliana RACK1 proteins (Figure 1A)
Summary
The receptor for activated C-kinase 1 (RACK1) is a conserved protein belonging to the WD40 repeat family of proteins It folds into a beta propeller with seven blades which allow interactions with many proteins. They are sensed by cell surface receptors which in case of G protein-coupled receptors interact with heterotrimeric guanine nucleotide binding proteins (G proteins), key intermediates in cellular signaling processes that link the receptors with intracellular effector proteins generating cellular responses [1,2]. The liberated Gβγ subunits play critical roles in many cellular processes [3,4] They regulate a variety of effector molecules ranging from enzymes, such as phospholipase Cβ (PLCβ) and adenylyl cyclase, to ion channels. D. discoideum cells lacking functional G protein β subunit are severely defective in phagocytosis, chemotaxis, aggregation, and development [17,12,18,19,20,21]
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