Mehta KD et al. (1991), The low density lipoprotein receptor in Xenopus...

XB-ART-24771

J Biol Chem 1991 Jun 05;26616:10406-14.

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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).

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Species referenced: Xenopus laevis
Genes referenced: apoe ldlr

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	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.