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The low density lipoprotein receptor in Xenopus laevis. I. Five domains that resemble the human receptor.
Mehta KD
,
Chen WJ
,
Goldstein JL
,
Brown MS
.
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All five functional domains of the low density lipoprotein (LDL) receptor were assembled in their modern form more than 350 million years ago, as revealed from the sequence of two cloned cDNAs from the frog Xenopus laevis. The two cDNAs appear to represent duplicated copies of the LDL receptor gene that arose when the entire genome of Xenopus duplicated approximately 30 million years ago. Both frog LDL receptors bound Xenopus LDL with high affinity and human LDL with lower affinity when expressed in monkey COS cells. The receptors also showed high affinity for rabbit beta-migrating very low density lipoprotein and canine apoE-HDLc, both of which contain apolipoprotein E. Each of the seven cysteine-rich repeats in the ligand binding domain of the Xenopus receptors resembles its counterpart in the human, indicating that these repeats had already acquired their independent structures by the time of amphibian development. The cytoplasmic tail of both Xenopus receptors is 86% identical to the human, including the FDNPVY sequence necessary for internalization in coated pits. The attainment of a fully developed receptor structure in Xenopus suggests that earlier forms of the receptor may exist in animals that are older than amphibians. An accompanying paper demonstrates that expression of both Xenopus receptor genes is controlled by a sterol regulatory element that closely resembles the human sequence (Mehta, K.D., Brown, M.S., Bilheimer, D.W., and Goldstein, J.L. (1991) J. Biol. Chem. 266, 10415-10419).
FIG. 1. Strategy for the generation of an oligonucleotide
probe specific for Xenopus LDL receptor. Oligonucleotide
primers correspondingt o the most conserved region of exon 9 of LDL
receptor from human, rabbit, and hamster were synthesized (shown
by filled boxes). Primer 1 was labeled with [yX2P]ATPa nd T4 kinase.
PCR was carried out using Xenopus genomic DNA, human genomic
DNA, and X. laeuis oocyte cDNA library as described under "Experimental
Procedures," and the reaction mixtures were subjected to
electrophoresis on a 10% polyacrylamide gel. The DNA sequences of
the PCR products of the major "P-labeled bands of 144 base pairs
(bp) were obtained as described under "Experimental Procedures."
The predicted amino acid sequence for the Xenopus PCR product is
shown.
FIG. 2. Nucleotide and deduced
amino acid sequence of Xenopus
LDL receptor cDNAs. The nucleotide
and amino acid sequence of LDL receptor
1 is shown. Nucleotide and amino
acids identical between LDL receptors 1
and 2 are shown as blank spaces whereas,
nucleotide substitutions are shown
above the receptor 1 nucleotide sequence,
and amino acid substitutions are
shown below the receptor 1 amino acid
sequence. The nucleotides are numbered
on the right side; nucleotide 1 is the A of
the initiator ATG codon; a negative
number refers to the 5'-untranslated region.
A portion of the receptor 1 5'-
untranslated region is shown in brackets;
the corresponding sequence for receptor
2 is not known. Amino acids are numbered
on the left; residue 1 is the alanine
found at theN H2 terminuso f the mature
human LDL receptor protein (3); negative
numbers refer to the cleaved signal
sequence. Amino acids that differ between
the two Xenopus LDL receptors
are boxed.
FIG. 3. Comparison of the amino
acid sequence of the human LDL
receptor with that of Xenopus LDL
receptor 1. Amino acids are numbered
on the left; residue 1 is the alanine found
at the NH, terminus of the mature human
LDL receptor protein (3). Identical
amino acids are boxed.
FIG. 4. Alignment of the amino
acid sequence of the ligand binding
domain of the LDL receptor from
human, rabbit, hamster, rat, and
Xenopus. Amino acids that occur at
least four times in a given repeat are
bored. Amino acids that occur at a given
position in at least 21 out of 42 interspecies
repeats are designated as consensus
residues. Frequency denotes the number
of occurrence of the consensus amino
acid in the 42 repeats analyzed. The
heavy underlines indicate "signature" sequences
that are uniquely characteristic
for each repeat.
FIG. 5. Blot hybridization (Northern analysis) of electrophoretically
separated RNA from Xenopus tissues. Poly(A)'
RNA (5 pg) from the indicated tissue was subjected to electrophoresis
on a 1.5% agarose gel as described under "Experimental Procedures"
and blotted onto a nylon membrane. Hybridization was carried out
at 37 "C for 16 h with a mixture of three single-stranded, uniformly
'"P-labeled Xenopus LDL receptor 1 cDNA probes of 150-200 nucleotides
in length (5 X lo6 cpm/ml). The primers for the three :'2P
probes corresponded to nucleotides 482-502, 1318-1338, and 2180-
2197 in pXLDLR-1. The primers were extended on an M13 template
in the presence of [a-'*P]dCTP, the products were subjected to
electrophoresis, and the bands corresponding to the longest products
were eluted from the gels and mixed together. After blotting, the
RNA-containing filter was exposed to Kodak XAR-5 film with an
intensifying screen for 5 h at -70 "C. The positions of RNA standards
run in an adjacent lane are indicated.
FIG. 6. Biosynthesis in transfected COS cells of Xenopus
LDL receptors 1 and 2. COS-M6 cells were transfected as described
under "Experimental Procedures" with 2 pgldish of one of the following
DNA preparations: lanes A and E, salmon sperm; lanes B and F,
pLDLR-17 encoding the human LDL receptor (11); lanes C and G,
pXLDLR-1; or lanes D and H, pXLDLR-2. Forty-eight hours after
transfection duplicate dishes from each transfection were pulse labeled
for 90 min with Trans"S"labe1. One dish from each set was
harvested for immunoprecipitation as described under "Experimental
Procedures." The remaining dish from each set was chased for 2 h in
medium containing 5 mM unlabeled methionine and cysteine and
then harvested for immunoprecipitation. The cell extracts in lanes B
and F were immunoprecipitated with anti-human LDL receptor IgG;
the cell extracts in all other lanes were immunoprecipitated with the
anti-Xenopus LDL receptor IgG. Immunoprecipitates were subjected
to SDS-gel electrophoresis and autoradiography. The dried gel was
exposed to XAR-5 film for 16 h at -70 "C. The positions to which
molecular weight marker proteins migrated in the gel are indicated
on the left.
FIG. 7. Saturation curves for the surface binding of Xenopus
'"I-LDL in COS cells transfected with a plasmid encoding
Xenopus LDL receptor 1 (A) or LDL receptor 2 (A). Monolayers
of COS-M6 cells were transfected with salmon sperm DNA (0) or
with an expression plasmid encoding either Xenopus LDL receptor 1
(pXLDLR-1) (A) or Xenopus LDL receptor 2 (pXLDLR-2) (A) as
described under "Experimental Procedures." Forty-eight hours after
transfection each monolayer was incubated for 3 h at 4 "C with the
indicated concentration of Xenopus "'I-LDL (665 cpm/ng protein),
after which the amount of suramin-releasable Y-LDL was determined.
FIG. 8. Saturation curves for the surface binding of Xenopus
and rabbit lipoproteins in COS cells transfected with a
plasmid encoding Xenopus LDL receptor 2. Monolayers of COSM6
cells were transfected with salmon sperm DNA (0) or an expression
plasmid encoding Xenopus LDL receptor 2 (pXLDLR-2) (A) as
described under "Experimental Procedures." Forty-eight hours after
transfection each monolayer was incubated for 3 h at 4 "C with the
indicated concentration of Xenopus lZ5I-LDL (556 cpm/ng protein)
( A ) or rabbit '251-p-VLDL (300 cpm/ng protein) (B), after which the
amount of suramin-releasable '2'I-ligand was determined.
FIG. 9. Saturation curves for the surface binding of canine
1261-apoE-HDLi,n COS cells transfected with a plasmid encoding
the human LDL receptor (0) or Xenopus LDL receptor
2 (A). Monolayers of COS-M6 cells were transfected with salmon
sperm DNA (0) or with an expression plasmid encoding either the
human LDL receptor (pLDLR-17) (0) (Ref. 11) or Xenopus LDL
receptor 2 (A) as described under "Experimental Procedures." Fortyeight
hours after transfection each monolayer was incubated for 3 h
at 4 "C with the indicated concentration of canine '251-apoE-HDL,
(113 cpm/ng protein), after which the amount of suramin-releasable
"'I-apoE-HDL, was determined.
FIG. 10. Competition by unlabeled canine apoE-HDL, (0)
and Xenopus LDL (0) for the cell surface binding of Xenopus
Iz6I-LDL in COS cells transfected with a plasmid encoding
Xenopus LDL receptor 2. Monolayers of COS-M6 cells were
transfected with pXLDLR-2 as described under "Experimental Procedures."
Forty-eight hours after transfection each monolayer was
incubated for 3 h at 4 "C with 5 pg of protein/ml of Xenopus '"I-LDL
(516 cpm/ng protein) in the presence of the indicated concentration
of unlabeled lipoprotein. After incubation for 3 h at 4 "C, the amount
of suramin-releasable "'I-LDL was determined. The 100% of control
value in the absence of unlabeled lipoprotein was 563 ng/mg protein.
FIG. 11. Competition by unlabeled ligands for the cell surface
binding of Xenopus lZ6I-LDL in COS cells transfected
with a plasmid encoding Xenopus LDL receptor 2. Monolayers
of COS-M6 cells were transfected with pXLDLR-2 as described under
"Experimental Procedures." Forty-eight hours aftetrr ansfection each
monolayer was incubated for 3 h at 4 "C with 5 pg of protein/ml of
Xenopus "'I-LDL (535 cpm/ng protein) in the absence (0) or presence
of the indicated concentrations of unlabeled ligand. After incubation
for 3 h at 4 "C, the amount of suramin-releasable "'I-LDL
was determined.