ARP IS A PLASMA MEMBRANE-ASSOCIATED RAS-RELATED GTPASE WITH REMOTE SIMILARITY TO THE FAMILY OF ADP-RIBOSYLATION FACTORS

The human and rat homologues of a novel Ras-related GTPase with unique structural features were cloned by polymerase chain reaction amplific ation and cDNA library screening. Their deduced amino acid sequences a re highly homologous (97% identical amino acids; 88.3% identical nucle otides within the coding region) and comprise all six of the conserved motifs presumably involved in GTP binding. Because the sequences exhi bit some similarity with members of the ADP-ribosylation factor (ARF) family (33% identity with ADP-ribosylation factor 1 (ARF1), 39% identi ty with ARF-like 3), the protein was designated ARP (ARF-related prote in). In contrast to all other members of the ARF family, ARP lacks the myristoylation site at position 2 and comprises an insertion of 8 ami no 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 mem branes from rat liver, testis, and kidney. Thus, the protein appears t o be posttranslationally modified for membrane anchoring. Unlike ARF, the ARP immunoreactivity was detected in plasma membranes but not in c ytosol of fractionated 3T3-L1 cells. Recombinant ARP exhibited specifi c and saturable GTP gamma S (guanosine 5'-3-O-(thio)triphosphate) bind ing and, unlike ARF isotypes, GTPase activity in the absence of tissue extracts or phospholipids. Thus, the structural and functional charac teristics of ARP indicate that it represents a novel subtype of GTPase s, presumably exerting a unique function and possibly involved in plas ma membrane-related signaling events.