Rab geranylgeranyl transferase alpha mutation in the gunmetal mouse reduces Rab prenylation and platelet synthesis

Abstract

Few molecular events important to platelet biogenesis have been identified. Mice homozygous for the spontaneous, recessive mutation gunmetal (gm) have prolonged bleeding, thrombocytopenia, and reduced platelet alpha- and delta-granule contents. Here we show by positional cloning that gm results from a G-->A substitution mutation in a splice acceptor site within the alpha-subunit of Rab geranylgeranyl transferase (Rabggta), an enzyme that attaches geranylgeranyl groups to Rab proteins. Most Rabggta mRNAs from gm tissues skipped exon 1 and lacked a start codon. Rabggta protein and Rab geranylgeranyl transferase (GGTase) activity were reduced 4-fold in gm platelets. Geranylgeranylation and membrane association of Rab27, a Rab GGTase substrate, were significantly decreased in gm platelets. These findings indicate that geranylgeranylation of Rab GTPases is critical for hemostasis. Rab GGTase inhibition may represent a new treatment for thrombocytosis and clotting disorders.

Figure 1

C57BL/6J-gm/gm mice (Left) have a lighter coat color than coisogenic C57BL/6J-+/+ mice (Right).

Figure 2

Genetic and physical maps of mouse chromosome 14 in the vicinity of gm. (A) Consensus genetic map of the gm region on chromosome 14 derived from three backcrosses (MGD J:52722). The circle represents the centromere. Distances between markers are in centimorgans (cM). Numbered loci represent microsatellite markers. Relative positions of loci were ascertained from 769 [(C57BL/6J-gm × SPRET)F₁ × C57BL/6J-gm], 727 [(C57BL/6J-gm × PWK)F₁ × C57BL/6J-gm], and 966 [(C57BL/6J-gm × DBA)F₁ × C57BL/6J-gm] backcross mice. No recombination was found between gm and Rabggta. Dotted lines link the consensus gm genetic map to the physical map. (B) Physical map of the gm nonrecombinant interval (MGD J:52722). The gm critical region is delineated by chromosome crossovers (denoted by red X). gm was flanked by D14Mit63 (proximal), and D14Mit153 and D14Mit122 (distal). YAC (blue lines) and BAC (red lines) clones are shown. Loci physically mapped within YAC and BAC clones are indicated with colored circles. The location of Rabggta is indicated by red circles. Green circles indicate genes near gm that (C) also map to the homologous human chromosomal segment (14q11.2).

Figure 3

A splice acceptor mutation in Rabggta causes abnormal splicing. (A) Genomic structure of the 5′ end of mouse Rabggta. Exon α encodes 5′ untranslated region. The initiation methionine (bold and underlined ATG) occurs at the 3′ end of exon 1. Rabggta nucleotide sequence above is C57BL/6J-+/+ and below is C57BL/6J-gm/gm. The splice acceptor mutation is indicated by *. Rabggta exons connected by solid green lines represent normal splicing. Dashed red lines indicate exon 1 skipping in gm/gm RNA, and dotted blue lines indicate cryptic splice sites used by gm/gm. (B) RT-PCR analysis of RNA from wild-type (+/+) and mutant (gm/gm) bone marrow. The Rabggta oligonucleotides used amplified the region between exon α and exon 2. Each band was excised and sequenced: Normal bone marrow (+/+) shows the expected product of 486 bp (band a) (GenBank accession no. AF127656), and a product (638 bp) retaining intron α (band b) (GenBank accession no. AF127658). gm/gm bone marrow shows three bands: Band c (429 bp) (GenBank accession no. AF127657) represents exon 1 skipping; band b′ (638 bp) (GenBank accession no. AF127659) retains intron α; band d (557 bp) (GenBank accession no. AF127662) represents utilization of cryptic splice sites in intron α and exon 1. (C) Sequence analysis of Rabggta genomic DNA. Arrows indicate the splice acceptor mutation: +/+ = G, gm/gm = A, gm/+ exhibits both nucleotides (G and A).

Figure 4

Decreased Rabggta expression and Rab GGTase activity in gm/gm mice. (A) Western blots of wild-type (+/+), heterozygous (gm/+), and mutant (gm/gm) platelets probed with Rabggta and β-actin antibodies. Rabggta (60 kDa) was reduced ≈70% in gm/gm. This experiment was repeated four times with the same results. (B) Rab GGTase activity of gm/gm and +/+ platelets. Duplicate cytosolic protein extracts from two samples of pooled platelets from gm (open symbols) and C57BL/6J mice (solid symbols) were assayed for Rab GGTase activity by measuring transfer of [³H]geranylgeranyl ([³H]GG) to Rab1a (15). This experiment was repeated four times with platelets, and twice with liver, spleen, and kidney protein extracts with similar results. (C) GGTase-I activity of gm/gm and +/+ pooled platelets. GGTase-I activity was determined by measuring transfer of [³H]GG to Rac1. Each point represents an average of duplicate reactions. For Rab GGTase reactions, a control reaction incubated after addition of 50 mM EDTA was subtracted from all samples. For GGTase-I reactions, a control reaction incubated with buffer alone was subtracted from all samples. These experiments were repeated at least twice in each of the liver, kidney, and spleen extracts with similar results to platelets.

Figure 5

Deficient prenylation of Rab27 in gm platelets. (A) Western blots of wild-type (+/+), heterozygous (gm/+), and mutant (gm/gm) platelets and bone marrow incubated with Rab27 and β-actin antibodies. This experiment was repeated five times with the same results. (B) Western blots of subcellular fractions of wild-type (+/+) and mutant (gm/gm) platelets incubated with radiolabeled GTP or Rab27 antibodies. The subcellular fractions were total protein (T), membrane-associated protein (M), and soluble-fraction protein (S). This experiment was repeated five times with similar results. (C) Photomicrographs (×200) of +/+ and gm/gm platelets stained with Rab27 antibodies and detected with a FITC-conjugated secondary antibody. This experiment was repeated three times with similar results.