Abstract
The human and rat homologues of a novel Ras-related GTPase with unique structural features were cloned by polymerase chain reaction amplification and cDNA library screening. Their deduced amino acid sequences are highly homologous (97% identical amino acids; 88.3% identical nucleotides within the coding region) and comprise all six of the conserved motifs presumably involved in GTP binding. Because the sequences exhibit some similarity with members of the ADP-ribosylation factor (ARF) family (33% identity with ADP-ribosylation factor 1 (ARF1), 39% identity with ARF-like 3), the protein was designated ARP (ARF-related protein). In contrast to all other members of the ARF family, ARP lacks the myristoylation site at position 2 and comprises an insertion of 8 amino acids in the region between PM1 and PM2. mRNA was found in most rat tissues examined (skeletal muscle, fat, liver, kidney, spleen, testis, adrenals, ovary, thymus, intestine, and lung). Western blot analysis with antiserum against recombinant ARP showed a 25-kDa protein in membranes from rat liver, testis, and kidney. Thus, the protein appears to be post-translationally modified for membrane anchoring. Unlike ARF, the ARP immunoreactivity was detected in plasma membranes but not in cytosol of fractionated 3T3-L1 cells. Recombinant ARP exhibited specific and saturable GTP gamma S (guanosine 5'-3-O-(thio)triphosphate) binding and, unlike ARF isotypes, GTPase activity in the absence of tissue extracts or phospholipids. Thus, the structural and functional characteristics of ARP indicate that it represents a novel subtype of GTPases, presumably exerting a unique function and possibly involved in plasma membrane-related signaling events.
Highlights
ADP-ribosylation factors (ARF)1 represent a subfamily of Ras-homologous GTPases (Kahn and Gilman, 1984; Bobak et al, 1989; Kahn et al, 1991) presumably involved in basic cellular functions, e.g. regulation of phospholipase D (Brown et al, 1993; Kahn et al, 1993; Cockcroft et al, 1994), exocrine secretion (Zeuzem et al, 1992b), vesicle traffic from ER to Golgi (Donaldson et al, 1991; Serafini et al, 1991; Kahn et al., The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) X78603 and X91504
All isoforms exhibit a high degree of overall structural similarity (65–96% identical amino acids) and share motifs determining common functional characteristics, e.g. the N-terminal myristoylation site
We describe the identification of a new GTPase which exhibits a remote similarity with other members of the extended ARF family
Summary
ADP-ribosylation factors (ARF) represent a subfamily of Ras-homologous GTPases (Kahn and Gilman, 1984; Bobak et al, 1989; Kahn et al, 1991) presumably involved in basic cellular functions, e.g. regulation of phospholipase D (Brown et al, 1993; Kahn et al, 1993; Cockcroft et al, 1994), exocrine secretion (Zeuzem et al, 1992b), vesicle traffic from ER to Golgi (Donaldson et al, 1991; Serafini et al, 1991; Kahn et al., The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) X78603 and X91504. Several genes encoding proteins with homology to the ARF isoforms have been cloned and designated ARF-like genes (Tamkun et al, 1991; Clark et al, 1993; Schurmann et al, 1994; Cavenagh et al, 1994) The products of these genes appear to lack ADP-ribosylation enhancing activity, and show different characteristics of GTP-binding (dependence on magnesium and phospholipids) than the ARF isoforms (Cavenagh et al, 1994). We describe the identification of a new GTPase which exhibits a remote similarity with other members of the extended ARF family Since it differed in important characteristics from other ARF and ARF-like proteins, e.g. in the lack of a myristoylation motif, in a constitutive GTPase activity, and in its subcellular distribution, the protein was designated ARF-related protein (ARP)
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