(Received for publication, June 22, 1995; and in revised form, July 20, 1995)
From the
The insulin-regulated glucose transporter isotype GluT4 expressed only in muscle and adipose cells is sequestered in a specific secretory vesicle. These vesicles harbor another major protein, referred to as vp165 (for vesicle protein of 165 kDa), that like GluT4 redistributes to the plasma membrane in response to insulin. We describe here the cloning of vp165 and show that it is a novel member of the family of zinc-dependent membrane aminopeptidases, with the typical large extracellular catalytic domain and single transmembrane domain but with a unique extended cytoplasmic domain. The latter contains two dileucine motifs, which may be critical for the specific trafficking of vp165, since this has been shown to be the case for this motif in GluT4. However, the tissue distribution of vp165 is much wider than that of GluT4; consequently, vp165 may also function in processes unrelated to insulin action and may serve as a ubiquitous marker for a specialized regulated secretory vesicle.
Adipose and muscle cells contain a specific type of glucose transporter GluT4(1) . As determined by immunoelectron microscopy, this isoform is almost exclusively found in intracellular vesicular and tubular structures and the trans-Golgi reticulum in untreated cells(2, 3) . Insulin treatment leads to the rapid redistribution of GluT4 from this intracellular pool to the cell surface and thus to a large increase in glucose transport (reviewed in (4) and (5) ). An enhanced rate of fusion of the GluT4-containing vesicles with the plasma membrane is most likely largely responsible for this effect(6) .
Unique GluT4 vesicles have been isolated from the low density microsomal fraction of adipocytes and skeletal muscle cells by immunoadsorption and by gel filtration chromatography and sucrose gradient centrifugation, and further characterized(3, 7, 8, 9, 10) . Members of the VAMP and SCAMP family, proteins first described in synaptic and secretory vesicles, respectively, were shown by immunoblotting to be concentrated in these vesicles(11, 12, 13, 14) . Moreover, several additional polypeptides have been found by protein staining, prominent among which is a protein of molecular mass 165 kDa (referred to herein as vp165 for vesicle protein of 165 kDa)(3, 7, 8, 9, 10) . Another group (Kandror and Pilch(7) , who designated this protein gp160) and ourselves (8) have purified vp165 from rat adipocytes and obtained the sequences of tryptic peptides. Through detection of vp165 with antibodies against two of these peptides, it has been shown that vp165 is concentrated in GluT4 vesicles in basal adipocytes and redistributes to the plasma membrane in response to insulin(8, 15) . Subsequently, while this study was in progress, Kandror et al.(16) provided indirect evidence that vp165 is an aminopeptidase. We now report the cloning of vp165 and its definitive characterization as a novel membrane aminopeptidase.
The 5`-end of the cDNA was obtained by 5`-RACE using the 5`-RACE system from Life Technologies, Inc. Total RNA (1 µg) purified from rat epididymal fat pads with Trisolv (Biotecx Laboratories, Inc.) was reverse transcribed with the degenerate antisense primer given above at 4 µM. The dCTP-tailed cDNA (2/5 of the total) was used in an amplification reaction with the specific primer 5`-AGGTTCAAACTGAGTTGCTGC-3` (nt 939-959) and the anchor primer under conditions optimal for the UlTma DNA polymerase, which has an inherent 3` to 5`-exonuclease proofreading activity (Perkin-Elmer Corp.). After an initial denaturation at 94 °C for 5 min and addition of the polymerase, 35 cycles were run with denaturation at 94 °C for 1 min, annealing at 57 °C for 1 min, and extension at 72 °C for 1 min 30 s, followed by a final extension at 72 °C for 10 min. The predominant PCR product at 1 kb was gel purified and reamplified with the nested primer 5`-ATCTGTGTAGGTGATGCCA-3` (nt 896-914) and the universal anchor primer. The PCR product was subcloned into pCR-Script (SK+) (Stratagene) and sequenced (nt 1-913).
The 3`-end
was obtained in two steps by the 3`-RACE method(17) . In the
first step a degenerate primer derived from one of the tryptic peptides
(residues 894-905), which was missing in the sequence so far
obtained, was attached to an adaptor
(5`-GGCCACGCGTCGACTAGTACTTRTGNGCRTCHGC-3`) and used at 2 µM to reverse transcribe 0.5 µg of poly(A) RNA
purified from isolated rat adipocytes using the FastTrack mRNA
isolation kit (Invitrogen). The cDNA purified with GlassMAX (Life
Technologies, Inc.) was amplified with the sense primer
5`-GAAATCCCTATGTTCTGAGTGACAA-3` (nt 2224-2248) and the adaptor
primer 5`-GGCCACGCGTCGACTAGTAC-3` under conditions used for the
amplification in the 5`-RACE, but with annealing at 60 °C and
extension at 72 °C for only 1 min. The predominant product at 600
bp was subcloned and sequenced (nt 2224-2771). The most 3`-end
was then obtained as described above except with an oligo(dT)-adaptor
primer used at 4 µM for the reverse transcription
(5`-GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTTTTT-3`). In the primary
amplification reaction, the sense primer 5`-CTGTGTTCAAAGTTGGAGCAAGA-3`
(nt 2656-2678) and the adaptor primer 5`-GGCCACGCGTCGACTAGTAC-3`
were used. A small aliquot (1/50) of the first amplification reaction
was reamplified with a nested sense primer 5`-GGCTGGTTGTTCCTCTTTAGC-3`
(nt 2688-2708) and the adaptor primer. The PCR conditions were as
described for the first 3`-RACE step. The major product at 500 bp was
subcloned and sequenced (nt 2688-3171). All cDNAs were sequenced
on both strands by the dideoxynucleotide method with the Sequenase Kit
(U. S. Biochemical Corp.). Sequence analyses were done with the
software provided in the BIONET Time Sharing Service from
IntelliGenetics Inc.
Figure 1:
Nucleotide sequence of the rat vp165
cDNA and deduced amino acid sequence. The numbers on the right of the sequences give the positions of the last
nucleotide or amino acid residue on each line. The first
consensus Kozak start site (aagatgg) (41) in the nucleotide
sequence, the stop signal (tag), and the potential polyadenylation
signal (aataca) (35) are underlined. Although upstream
of the indicated Kozak start site there is no in-frame stop signal,
translation most likely initiates here, since the mouse cDNA, otherwise
identical to the rat sequence in this region, contains an additional G
after nucleotide 66, which results in a completely different predicted
amino acid sequence upstream of this methionine ( S. A. Ross and S. R.
Keller, unpublished data). In the amino acid sequence, the four tryptic
peptides obtained from the purified protein(8) , as well as the
two dileucines in the cytoplasmic domain and the zinc-binding domain
(IIAHELAHQWFG and LWLNEGF), are underlined. The potential
transmembrane domain is doubleunderlined. The 16
potential extracellular N-linked glycosylation sites are
marked with an asterisk. The beginning and the end of the
domain most conserved between vp165 and other aminopeptidases are
marked with and
, respectively (for alignment, see Fig. 3).
Figure 2:
Topology of vp165. a,
hydrophobicity profile of the deduced amino acid sequence for vp165.
The sequence was analyzed with the TopPred II software(42) . b, protease treatment. LDM were treated with proteinase K for
indicated times in the absence(-) or presence (+) of the
nonionic detergent CE
and immunoblotted with
antibodies against the N terminus (left) and C terminus (right) of vp165. Molecular mass markers in kDa are indicated
on the right of the blots.
Figure 3: Comparison of vp165 with homologous aminopeptidases. a, schematic illustration of the overall structure for vp165, aminopeptidase N from rat (24) (AMPNrat), aminopeptidase A from human (23) (AMPAhuman), thyrotropin-releasing hormone degrading enzyme from rat (27) (TRHDErat), aminopeptidase yscII from S. cerevisiae(25) (yscIIyeast), and aminopeptidase N from L. lactis(26) (AMPNlacto). The arrows define the location of the zinc-binding motif in all of the sequences and the two unique double leucines in vp165. The lengths of the boxes are proportional to the lengths of the amino acid sequences. b, alignment of the amino acid sequences in the region most conserved between the aminopeptidases. Abbreviations are as stated above. The numbers on the left of each line give the position of the first amino acid residue in the respective sequences. The alignment was initially done with GENALIGN (BIONET Time Sharing Service, IntelliGenetics Inc.) and was subsequently maximized by eye. Gaps (indicated by a dot) have been introduced for optimal alignment. Amino acid residues identical for at least four of the aligned sequences are boxed in black, and amino acid residues conserved between at least 4 of the aligned sequences are in gray. The numbers at the end of each sequence indicate the % identity of the respective sequence with vp165.
The predicted 105-kDa molecular mass for vp165 is less than the apparent 165-kDa size of the protein on SDS-polyacrylamide gels. This difference is at least in part due to glycosylation of the protein. The amino acid sequence contains 16 potential extracellular N-glycosylation sites (marked with an asterisk in Fig. 1), and treatment of GluT4 vesicle proteins with peptide N-glycosidase F caused a decrease in the mass of vp165 to 140 kDa (data not shown).
Because of this similarity,
we assayed detergent-solubilized GluT4 vesicles for aminopeptidase
activity using as substrates the aminoacyl -naphthylamides derived
from all 20 amino acids, except cysteine. The leucine substrate was
hydrolyzed most efficiently, followed by lysine at 58%, methionine at
44%, alanine at 28%, and arginine at 23% of the rate for leucine; the
other substrates were cleaved at less than 7% of the rate for leucine ( Table 1and data not shown).
To determine whether the vesicular
aminopeptidase activity was due to vp165, we immunoprecipitated vp165
from detergent-solubilized GluT4 vesicles with an antibody against its
N terminus and assayed the immunoprecipitate and the depleted
supernatant for activity against aminoacyl -naphthylamide
substrates (Table 1). For each substrate, approximately 85% of
the total activity associated with GluT4 vesicles was recovered in the
vp165 immunoprecipitate. The activities in the immunoprecipitates and
depleted supernatants correlated with the distribution of vp165 protein
as assessed by immunoblotting the same samples for vp165 (data not
shown). From these data, we conclude that vp165 is an aminopeptidase
and that it accounts for all the aminopeptidase activity found in the
GluT4 vesicles. Its substrate preference is distinct from the mammalian
aminopeptidases A and N, which are most active against glutamyl and
alanyl substrates, respectively(29) .
Apart from the two tyrosines, there are other potential sites for
regulation by phosphorylation present in the cytoplasmic tail of vp165.
Several serines and threonines are found in recognition motifs for
protein kinase A (positions 51, 86, 91, 98), protein kinase C
(positions 51, 73, 86, 91, 98, 107), casein kinase I (positions 5, 19,
73), and casein kinase II (position 19)(33) . GluT4 has only
one major site of phosphorylation in vivo; this is serine 488,
which is adjacent to its dileucine (see above) and is in motifs
specific for both protein kinase A and casein kinase II. Although the
extent of phosphorylation in GluT4 in the plasma membrane decreases in
response to insulin and increases upon stimulation with a
-adrenergic agent, the functional significance of these changes is
not known(34) .
Figure 4:
Tissue distribution of vp165 mRNA and
protein. a, Northern blot. Poly(A) RNA from
various tissues (2 µg for each) was probed with a single-stranded
digoxigenin-11-dUTP-labeled PCR product corresponding to nucleotides
2224-2783 in vp165 cDNA. RNA markers in kb are indicated on the right. b, immunoblot. vp165 immunoprecipitates
derived from 375 µg of total protein for each tissue were blotted
with the antibody raised against the N-terminal domain of vp165. As a
control, the precipitate from heart with unspecific rabbit IgG is shown
(Heart IgG). Molecular mass markers in kDa are indicated on the right.
The size of the vp165 mRNA was approximately 12.5 kb, which is much larger than the cloned cDNA (3.2 kb). This result was unexpected, since the cDNA ends in a poly(A) sequence. A likely explanation is that there is a less abundant, shorter mRNA that gave rise to the oligo(dT)-primed cDNA. Consistent with this hypothesis is the fact that 22 bp upstream from the poly(A) tail in the cDNA there is an inefficiently used polyadenylation signal, AATACA(35) . The size of the vp165 protein was 165 kDa in all tissues, except brain, where it was 140 kDa. Most likely, this is due to a difference in glycosylation of the brain protein.
The tissue distribution of vp165 differs markedly from that of GluT4, which is restricted to skeletal muscle, heart, and white and brown adipose tissue(1) . Thus, even though vp165 is localized in GluT4 vesicles in rat adipocytes(8, 15) , it is not specific to these insulin-regulated vesicles. It has recently been hypothesized that many cell types contain a unique intracellular storage vesicle involved in transient remodeling of the cell surface in response to specific stimuli(36) . If so, vp165 may be a widely distributed marker for this regulated secretory vesicle.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U32990[GenBank].