Proc. Natl. Acad. Sci. USA Vol. 91, pp. 9725-9729, October 1994 Medical Sciences Complete cloning and sequencing of rat gp330/"megalin," a distinctive member of the low density lipoprotein receptor gene family (endocytosis receptor/glomerular antgen/Heymann nephrits/membranous nephropathy/kidney proximal tubule) AKIHIKO SAITO*, SALVATORE PIETROMONACOt, ALVIN KWOR-CHIEH Loo*, AND MARILYN GIST FARQUHAR*t *Division of Cellular and Molecular Medicine and tThe Center for Molecular Genetics, University of California, San Diego, La Jolla, CA 92093-0651; and tDepartment of Medicine, University of New Mexico Cancer Center, Albuquerque, NM 87131-0001 Contributed by Marilyn Gist Farquhar, June 24, 1994 ABSTRACT We completed the cDNA cloning and sequenc- ing of gp330, the major kidney glomerular antigen for rat Heymann nephritis. The deduced 4660-aa sequence, expected to constitute a mature protein ofMr 516,715, consists of a probable N-terminal sinal peptide sequence (25 aa), an extracellular region (4400 aa), a single smembrane domain (22 aa), and a C-terminal cytoplasmic tail (213 aa). The extraceilular region contains three types of cysteine-rich repeats characteristic of the low density lipoprotein receptor (LDLR) gene family- 36 LDLR lignd-binding repeats forming four dusters of putative ligand-binding domains, 16 growth factor repeats separated by 8 YWTD spacer regions, and 1 C-terminal epidermal growth factor repeat. The cytoplasmic tail contains two copies of the (FX)NPXY motif, which represents a signal for coated pit- mediated inrlizatio and an additional similar motif. The overail structure of gp330 is similar to that of the LDLR-related protein (LRP)/a2-macrogiobulin receptor and shows even greater similait to the Caenorhabditis elegans protein, re- ported as a homologue of LRP. However, gp330 differs from these proteins in (i) the cysteine-rich repeat arrangements found in the extreme extraceilular N- and C-terminal regions, (ii) the distribution pattern of cysteine residues in the YWTD spacer regions, (Mi") the location of the RX(K/R)R consensus recogni- tion sequence of furin, a precursor processing endoprotease, and (iv) the length and rutrof the cytoplasmic tail. We suggest the name megaliln (from Greek mega) for gp330, the largest plasma membrane proti Identified so far in vertebrates. The cloned cDNA will be useful for studies on the physiological functions of gp330/megalin and for determining its role in Heymann nephritis. gp330 is a membrane glycoprotein originally identified as the major antigen involved in Heymann nephritis (HN) (1, 2), a rat model of membranous nephropathy in humans, caused by the binding of circulating antibodies to kidney glomeruli. gp330 is expressed in clathrin-coated pits on the plasma membrane of epithelial cells of the kidney glomerulus and proximal tubule (2, 3), lung (type II pneumocytes), epidid- ymis, and yolk sac, among others (4, 5). It forms a multimeric complex with another protein, referred to as the receptor- associated protein (RAP), in the endoplasmic reticulum (6-8) and at the cell surface (8, 9). Partial cDNA cloning (10) indicated that gp330 belongs to the low density lipoprotein receptor (LDLR) gene family (11, 12), which also includes the LDLR-related protein (LRP)/a2-macroglobulin receptor (13, 14), the very low density lipoprotein receptor (VLDLR) (15), the vitellogenin receptor (16), and a Caenorhabditis elegans protein (17). The precise biological functions of gp330 are still unclear; however, in vitro it has been shown to bind multiple ligands, including plasminogen (18), extracellular matrix components (19), plasminogen activator-plasminogen activator inhibitor type I complexes (20, 21), apolipoprotein E-enriched 3-VLDL (20), lipoprotein lipase (20, 22), lactoferrin (20), and Ca2+ (23). Many of these ligands as well as RAP are also bound by LRP (20). The similarities and differences between gp330 and LRP remain uncertain due to lack of information on the overall structure of gp330. In the current study, we present the complete primary structure of rat gp330 deduced from the cDNA sequence.§ The 4660-aa sequence is expected to constitute a mature protein with a Mr of 516,715, excluding glycosylation. The overall structure of gp330 and several other members of the LDLR gene family are very similar; however, some impor- tant differences are noted that suggest unique physiological and pathological roles for gp330. MATERIALS AND METHODS Isolation and Blotting of RNA. Total cellular RNA was isolated as described (24, 25) from adult Sprague-Dawley rat kidney, heart, lung, liver, brain, and skeletal muscle. Poly(A) RNA was selected from the kidney RNA with Oligotex-dT latex particles (Qiagen, Chatsworth, CA) for Northern blot analysis and cDNA library construction. Twenty micrograms of kidney poly(A) RNA and 0.65 jg of total cellular RNAs were blotted for Northern and slot blot analysis, respectively, to MagnaGraph nylon transfer membranes (Micron Separa- tions, Westboro, MA) and hybridized with 32P-labeled probes as described (25). Rat (3-actin cDNA was used as an internal control probe for slot blot analysis. Screening of cDNA Libraries. A rat kidney cDNA library in Agtll (Clontech) was screened as described (26) with anti- serum raised against high molecular weight, negatively charged antigens purified from rat glomeruli by size-ex- clusion and ion-exchange chromatography (27). The cDNA inserts of positive clones were excised and subcloned into the Bluescript vector (Stratagene) for characterization by restric- tion digestion and sequencing. A clone, ClB, was obtained by immunoscreening and used as a 32P-labeled probe to screen the Agtll library as described (25). Random- and oligo(dT)-primed cDNAs were synthesized and size selected by using poly(A) rat kidney RNA with a Superscript cDNA synthesis kit (GIBCO/BRL). The cDNAs were introduced into the EcoRI site of the AZAP II vector to construct libraries Abbreviations: HN, Heymann nephritis; LDLR, low density lipo- protein receptor; LRP, low density lipoprotein receptor-related protein; RAP, receptor-associated protein; VLDLR, very low den- sity lipoprotein receptor. §The sequence reported in this paper has been deposited in the GenBank data base (accession no. L34049). 9725 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Downloaded by guest on January 2, 2020
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Proc. Natl. Acad. Sci. USAVol. 91, pp. 9725-9729, October 1994Medical Sciences
Complete cloning and sequencing of rat gp330/"megalin," adistinctive member of the low density lipoprotein receptorgene family
AKIHIKO SAITO*, SALVATORE PIETROMONACOt, ALVIN KWOR-CHIEH Loo*, AND MARILYN GIST FARQUHAR*t*Division of Cellular and Molecular Medicine and tThe Center for Molecular Genetics, University of California, San Diego, La Jolla, CA 92093-0651; andtDepartment of Medicine, University of New Mexico Cancer Center, Albuquerque, NM 87131-0001
Contributed by Marilyn Gist Farquhar, June 24, 1994
ABSTRACT We completed the cDNA cloning and sequenc-ing of gp330, the major kidney glomerular antigen for ratHeymann nephritis. The deduced 4660-aa sequence, expected toconstitute a mature protein ofMr 516,715, consists ofa probableN-terminal sinal peptide sequence (25 aa), an extracellularregion (4400 aa), a single smembrane domain (22 aa), anda C-terminal cytoplasmic tail (213 aa). The extraceilular regioncontains three types of cysteine-rich repeats characteristic ofthelow density lipoprotein receptor (LDLR) gene family- 36LDLR lignd-binding repeats forming four dusters of putativeligand-binding domains, 16 growth factor repeats separated by8 YWTD spacer regions, and 1 C-terminal epidermal growthfactor repeat. The cytoplasmic tail contains two copies of the(FX)NPXY motif, which represents a signal for coated pit-mediated inrlizatio and an additional similar motif. Theoverail structure of gp330 is similar to that of the LDLR-relatedprotein (LRP)/a2-macrogiobulin receptor and shows evengreater similait to the Caenorhabditis elegans protein, re-ported as a homologue of LRP. However, gp330 differs fromthese proteins in (i) the cysteine-rich repeat arrangements foundin the extreme extraceilular N- and C-terminal regions, (ii) thedistribution pattern of cysteine residues in the YWTD spacerregions, (Mi") the location of the RX(K/R)R consensus recogni-tion sequence offurin, a precursor processing endoprotease, and(iv) the length and rutrof the cytoplasmic tail. We suggestthe name megaliln (from Greek mega) for gp330, the largestplasma membrane proti Identified so far in vertebrates. Thecloned cDNA will be useful for studies on the physiologicalfunctions of gp330/megalin and for determining its role inHeymann nephritis.
gp330 is a membrane glycoprotein originally identified as themajor antigen involved in Heymann nephritis (HN) (1, 2), arat model ofmembranous nephropathy in humans, caused bythe binding of circulating antibodies to kidney glomeruli.gp330 is expressed in clathrin-coated pits on the plasmamembrane of epithelial cells of the kidney glomerulus andproximal tubule (2, 3), lung (type II pneumocytes), epidid-ymis, and yolk sac, among others (4, 5). It forms a multimericcomplex with another protein, referred to as the receptor-associated protein (RAP), in the endoplasmic reticulum (6-8)and at the cell surface (8, 9). Partial cDNA cloning (10)indicated that gp330 belongs to the low density lipoproteinreceptor (LDLR) gene family (11, 12), which also includes theLDLR-related protein (LRP)/a2-macroglobulin receptor (13,14), the very low density lipoprotein receptor (VLDLR) (15),the vitellogenin receptor (16), and a Caenorhabditis elegansprotein (17).
The precise biological functions of gp330 are still unclear;however, in vitro it has been shown to bind multiple ligands,including plasminogen (18), extracellular matrix components(19), plasminogen activator-plasminogen activator inhibitortype I complexes (20, 21), apolipoprotein E-enriched3-VLDL (20), lipoprotein lipase (20, 22), lactoferrin (20), andCa2+ (23). Many of these ligands as well as RAP are alsobound by LRP (20). The similarities and differences betweengp330 and LRP remain uncertain due to lack of informationon the overall structure of gp330.
In the current study, we present the complete primarystructure of rat gp330 deduced from the cDNA sequence.§The 4660-aa sequence is expected to constitute a matureprotein with a Mr of 516,715, excluding glycosylation. Theoverall structure of gp330 and several other members of theLDLR gene family are very similar; however, some impor-tant differences are noted that suggest unique physiologicaland pathological roles for gp330.
MATERIALS AND METHODSIsolation and Blotting of RNA. Total cellular RNA was
isolated as described (24, 25) from adult Sprague-Dawley ratkidney, heart, lung, liver, brain, and skeletal muscle. Poly(A)RNA was selected from the kidney RNA with Oligotex-dTlatex particles (Qiagen, Chatsworth, CA) for Northern blotanalysis andcDNA library construction. Twenty microgramsof kidney poly(A) RNA and 0.65 jg of total cellular RNAswere blotted for Northern and slot blot analysis, respectively,to MagnaGraph nylon transfer membranes (Micron Separa-tions, Westboro, MA) and hybridized with 32P-labeled probesas described (25). Rat (3-actin cDNA was used as an internalcontrol probe for slot blot analysis.
Screening ofcDNA Libraries. A rat kidney cDNA library inAgtll (Clontech) was screened as described (26) with anti-serum raised against high molecular weight, negativelycharged antigens purified from rat glomeruli by size-ex-clusion and ion-exchange chromatography (27). The cDNAinserts ofpositive clones were excised and subcloned into theBluescript vector (Stratagene) for characterization by restric-tion digestion and sequencing. A clone, ClB, was obtainedby immunoscreening and used as a 32P-labeled probe toscreen the Agtll library as described (25). Random- andoligo(dT)-primed cDNAs were synthesized and size selectedby using poly(A) rat kidney RNA with a Superscript cDNAsynthesis kit (GIBCO/BRL). The cDNAs were introducedinto the EcoRI site ofthe AZAP II vector to construct libraries
Abbreviations: HN, Heymann nephritis; LDLR, low density lipo-protein receptor; LRP, low density lipoprotein receptor-relatedprotein; RAP, receptor-associated protein; VLDLR, very low den-sity lipoprotein receptor.§The sequence reported in this paper has been deposited in theGenBank data base (accession no. L34049).
9725
The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.
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with a AZAP I/EcoRI/Gigapack II cloning kit (Stratagene).The cDNA libraries were screened by hybridization withprobes to obtain overlapping clones. Positive clones were
recovered in the Bluescript vector using Stratagene's in vivoexcision protocol for further characterization.DNA Sequencing. Clones containing large inserts were
sequenced by the dideoxynucleotide chain-terminationmethod with Sequenase (United States Biochemical). Theclones were also digested with EcoRI and resulting insertfragments were subcloned into the Bluescript vector forsequencing. Multiple clones were sequenced on both strandsto confirm the sequence. Amino acid sequence deduced fromthe cDNA sequence was analyzed as described (28).
RESULTS
Cloning ofthe gp330 cDNA. By screening a rat kidney AgtllcDNA library with antiserum raised against large, negativelycharged antigens isolated from rat glomeruli, we identified a0.4-kb cDNA clone, C1B, which encodes a portion of gp330(10). The antiserum was immunopurified on the C1B/,-galactosidase fusion protein and used for immunoblot anal-ysis ofrat kidney microvillar membranes. Antibodies specificfor the CiB fusion protein reacted with a high molecularweight protein, which was identified as gp330 by using amonoclonal anti-gp330 antibody, thus confirming that CiBencodes gp330 (data not shown).The C1B cDNA was then used as a probe to screen the
Agtll cDNA library and five additional overlapping cloneswere obtained. The longest 2.1-kb clone, C1BP (Fig. 1), wasused as a probe to screen 1 x 105 recombinants in therandom-primed rat kidney AZAP II cDNA library. Twentypositive clones, which contained cDNA inserts >4 kb, wereidentified and used to construct restriction maps. A 1.3-kbPst I/EcoRI fragment was prepared from the 3' end of clone83 (Fig. 1) for use as a probe for the second screening, whichled to the identification of 14 overlapping clones. A 3.4-kbEcoRI fragment was prepared from clone 217 (Fig. 1) andused for screening the oligo(dT)-primed cDNA library toobtain the 3' end of the gp330 cDNA. Two of the clonesobtained, clones 306 and 313 (Fig. 1), which appeared tocontain the longest sequence of the 3' untranslated region,were sequenced.The Primary Structure of gp330. The assembled 15.4-kb
cDNA revealed an uninterrupted open reading frame en-coding 4660 aa (Fig. 2). The deduced amino acids contain allthe partial sequences published previously (10) except forfive single substitutions: Asn1075 (in Fig. 2) vs. Lys, Asn1325vs. Arg, Pro3789 vs. Ala, Ser397 vs. Leu, and Phe4576 vs. Ile,which may be due to polymorphic nucleotide changes. Thegp330 amino acid sequence predicts a type I transmembraneprotein consisting of a 25-aa probable N-terminal signalpeptide sequence (29), a 4400-aa extracellular region, a22-aa single transmembrane domain, and a 213-aa C-termi-nal cytoplasmic tail. The extracellular region is composedof three types of cysteine-rich repeat motifs-the LDLRligand-binding repeat (class A motif), the epidermal growthfactor repeat (class B.1 motif), and the growth factor repeat
ASPGADEIQGKKWNIFKRKPKQTTDWEIEMPLjEMDSEVKDAVAVAPPPSPSLPAKASKRNLTPGYTATEDTFKDTANLVKEDSDV 4635FIG. 2. Amino acid sequence of gp330. The probable signal
peptide sequence is underlined. The presumptive membrane-spanning sequence is indicated by double underlines. The two(FX)NPXY signals for coated pit-mediated internalization and anadditional similar motif [(VE)NQNY] in the cytoplasmic tail areboxed. The RX(K/R)R sequence, which might function as a cleavagesignal, is also boxed. Possible attachment sites for N-linked oligo-saccharides are indicated in boldface. Amino acids are numberedfrom the probable cleavage site of signal peptidase.
Proc. Natl. Acad Sci. USA 91 (1994)
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I II
Ni. il-wm111. 45/c. 35Fc 49%
2 26%c 48%
3 36'3 51%
Proc. Natl. Acad. Sci. USA 91 (1994) 9727
33% 44 41 ~ 1
28% 4 4(1t
--- usell35%l^( is t;; m.465
[ ligand-binding repeat (class A motif) * 0-linked sugar domaintralsmemralz) :I ~ll a l11)
-_.-.. YWTD spacer region (- cysteine) t R-X-K/R-R sequeIce
U 1 LL1:-T(.) . )
I',' I 1 RI1 \
FiG. 3. Schematic representation of protein structures of the major known members of the LDLR gene family. Amino acid identitypercentages are shown between the structurally conserved domains of rat gp330 and the C. elegans protein (row 1), between the C. elegansprotein and human LRP (row 2), between LRP and gp330 (row 3), as well as between human LDLR and VLDLR (row 4). Four putativeligand-binding domains of gp330 are indicated (I-IV). EGF, epidermal growth factor.
(class B.2 motif) (13). Each consists of -4O aa containing 6cysteine residues. The entire extracellular region is made upof 36 class A motifs of putative ligand-binding domainsarranged in four clusters, 16 class B.2 motifs separated by8 spacer regions containing the YWTD consensus se-quences, and 1 class B.1 motif following the last class B.2motif (Fig. 3). The SDE motif between the fifth and sixth
TRCNQ-LQFT-CL-----NGHCINQDWKCDNDNDCG--DGSDE----LPTVCT-CRS-TAFT-CG-----NGRCVPYHYRCDYYNDCG--DNSDE------AGCRNCNSTTEFT-CS-----NGRCIPLSYVCNGINNCHDNDTSDE------KNCT-CPP--DFTKCQT----TNICVPRAFLCDGDNDCG--DGSDE----NPIYCT-CRS-NEFQ-CLS----PQRCIPSYWFCDGEADCA--DGSDE-----PDTCT-CRA-SQFQ-CD-----NGRCISGNWVCDGDNDCG--DMSDE---DQRHHCQNCSS-TQFT-CVNSRPPNRRCIPQYWVCDGDADCS--DALDE-----LQNCT-CSA-GEFS-CA-----NGRCVRQSFRCDRRNDCG--DYSDE------RGCPPCHA-NQFT-CQ-----NGRCIPRFFVCDEDNDCG--DGSDE----QEHLCT-CPL-HQFR-CD-----NGHCIEMGRVCNHVDDCS--DNSDE------KGCT C QFT C NGRCIP VCDG NDCG D =DE C
C F C N CI CDG DC DG=EE CC F C NG C W CDG DC D =ES C
gp330 C F C N CIP W CDG DC DSflE CCelP C F C CIP CDG DCG DSD CLRP C QF C N RCIP W CDG DDCG DG D= E C
FIG. 4. Alignment of the 36 class A motifs in the four putativeligand-binding domains ofgp330. The consensus amino acids, whichoccur at a given position >50%1 of the time in each domain of ratgp330, the C. elegans protein (CeiP), and human LRP, are shownbelow. The highly conserved SDE motifs are underlined; lowercases in domain I notes the lower frequency. Amino acids are nuniberedfrom the probable cleavage sites of signal peptidase.
cysteines of the class A motifs [known to be important forhigh-affinity binding of positively charged sequences in theLDLR's ligands (12)] is highly conserved, although itdegenerates somewhat in the N-terminal putative ligand-binding domain (Fig. 4). gp330 contains one RX(K/R)R(RLRR) sequence in a YWTD spacer region (Figs. 2 and 3)that might be recognized by furin, a precursor processingendoprotease (12). The YWTD spacer regions contain 10cysteine residues that are not associated with the class Aand B motifs (Fig. 3). The cytoplasmic tail (Fig. 5A)contains two copies of the (FX)NPXY motif(FENPMY andFENPIY), which represents a signal for coated pit-mediated internalization (30, 31) and another similar motif,VENQNY. The cytoplasmic tail (213 aa) is much longerthan that of other members of the LDLR gene family andshows little sequence similarity to them except for the(FX)NPXY motifs and the extreme C-terminal end of thecytoplasmic tail where 4 out of the last 5 amino acids areidentical in sequence to the same region of the C. elegansprotein (17) (Fig. SB).RNA Expression. Northern blot analysis using C1BP as a
probe detected a major =15-kb band in rat kidney poly(A)RNA (Fig. 6A). RNA slot blot analysis was carried out usinga 1.2-kb EcoRI-digested fragment of clone 306 that encodesthe cytoplasmnic tail and the 3' untranslated region with no
V G K Q P V IF EQVTVP ENVEKP KQTTN[EFHGNVI SIFISMHDD S TT FTE KAD P LRVDNGAMNVE IGD PDKP TN[FTKNMK S MN F DKNINS IN FD
NPMrYAAKDNTSKVNQ NIYGRPIDPSEINP I YAEMDSEVKDNPVLENKQDAPGSNPV ELEDVDMSSNPLYIDPDSEVSDVNP TIYIKMYEGGEPDNPVIYIATLYMGGHGNP VIYILKTTEEDLSNP VMYQKTTEDEVH
NLVKW1D TV1DPDSJV|SDVEDE I G L
VVSTD DDLAM VSLE[DFVA
FIG. 5. Alignment of the cytoplasmic tail amino acid sequencesof the LDLR gene family. The only regions of gp330 that showsignificant sequence homology to other members of this family arethe (FX)NPXY motifs (A) and the extreme C-terminal5 amino acids,which are very similar to those of the C. elegans protein but not tothe others (B). CeIP, C. elegans protein; LRP, VLDLR, and LDLRare sequences from the human proteins. Consensus amino acidsoccurring >50%o of the time are boxed.
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B
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FIG. 6. RNA blots of gp330. (A) Northern blot of poly(A) RNA(20 pg) from rat kidney with C1BP as a probe. The arrowheadindicates the electrophoretic origin. (B) Slot blots of total RNA (0.65Mig each) from rat kidney (K), lung (Lu), skeletal muscle (S), brain(B), heart (H), and liver (Li) with a 1.2-kb EcoRI fragment of clone306 (lane 1) and rat ,B-actin cDNA (lane 2).
homology to any other members of the LDLR gene family.Compared with f-actin expression, the level of expression ofgp330 is high in kidney, less in lung, and low in liver, brain,heart, and skeletal muscle (Fig. 6B).
DISCUSSIONWe have completed the cDNA cloning and sequencing of themajor HN antigen, which has been referred to up until nowas gp330. The deduced amino acid sequence of rat gp330consists of 4660 residues with a predicted Mr of 516,715excluding glycosylation. Originally, the size of gp330 wasestimated to be 330 kDa (1, 2), but more- recently (6) it hasbeen estimated to be -500 kDa by SDS/PAGE. Since thename gp330 is no longer entirely appropriate, we suggest theterm megalin [from Greek mega (large)] for this protein,which is the largest plasma membrane protein identified sofar in vertebrates.The arrangements of the extracellular repeat motifs of rat
gp330/megalin and human (as well as mouse and chicken)LRP (12, 13) are well conserved in most regions (Fig. 3),which is in keeping with data indicating that gp330/megalinand LRP bind common ligands (20). However, the extracel-lular structures of these two LDLR gene family membersdiffer significantly in two regions: (i) in the N-terminalputative ligand-binding domain where rat gp330/megalin hasseven class A motifs while LRP has only two, and (ii) in theC-terminal region, where gp330/megalin has only one classB.1 motif while LRP has five (Fig. 3). These structuraldifferences presumably reflect functional differences be-tween these two molecules. Interestingly, the repeat motifstructure of rat gp330/megalin is more similar to that of theC. elegans protein, originally reported as a LRP equivalent(17), than to LRP. Unlike rat gp330/megalin, however, the C.elegans protein has six class A motifs in the N-terminalputative ligand-binding domain (instead of seven), which mayalso reflect unique binding functions as is the case for LDLRand VLDLR, which bind different ligands (Fig. 3). The classB.1 motifs in rat gp330/megalin and the C. elegans proteinare more similar in structure to one another than to those ofLRP (data not shown).The cytoplasmic tail of rat gp330/megalin (213 aa) is longer
than that of the C. elegans protein (157 aa) (17), human LRP(100 aa) (12, 13), human LDLR (50 aa) (32), or humanVLDLR (54 aa) (15). The larger size of the cytoplasmic tailmay influence the protein's structure, its intracellular traf-ficking, and/or its potential interaction with intracellularmessengers. The cytoplasmic tails of both rat gp330/megalinand the C. elegans protein contain two copies of the (FX)NPXY motif, which is a signal for coated pit-mediatedinternalization (30, 31), and one additional similar motif,
(VE)NQNY and (FS)NPVL, respectively (Fig. 5A). The(FX)NPXY motif appears only once in LDLR and VLDLRand twice in LRP (Fig. 5A). It is of interest that the extremeC-terminal sequence of the cytoplasmic tails of gp330/megafln and the C. elegans protein are more closely related,whereas those of LDLR, VLDLR, and LRP are closelyrelated (Fig. 5B), suggesting that these groups may undergodifferent intracellular trafficking and/or signaling pathwaysconsonant with different functions.LRP undergoes intracellular proteolytic processing (to 515-
and 85-kDa peptides) in a trans-Golgi compartment (33). Itscleavage site conforms to the RX(K/R)R consensus recog-nition sequence of furin, a precursor processing endopro-tease in the secretory pathway (12). Rat gp330/megalin andthe C. elegans protein (17) contain similar sequences (RLRRand RKKR, respectively) in the YWTD spacer region, butthey are in a different location (Figs. 2 and 3). There is noevidence that gp330/megalin is cleaved intracellularly (7, 8).However, it may be cleaved at the cell surface, because asoluble form has been found in the medium of culturedepithelia synthesizing gp330/megalin (9) and in human urine(22). Moreover, cleavage of gp330/megalin plays a key rolein the pathogenesis of HN; within 15 min after antibodybinding gp330/megalin is shed from the cell surface, andgp330-anti-gp330 complexes become attached to the glomer-ular basement membrane (3), thus initiating the formation ofimmune deposits. gp330/megalin cannot be detected in im-mune deposits with an antibody specific to a 17-aa sequencein the cytoplasmic tail (34).Another difference between the members of the LDLR
gene family is in the number and locations of cysteineresidues in the YWTD spacer regions. There are 1, 10, 19, and13 cysteine residues associated with these regions in humanLDLR (32), rat gp330/megalin, the C. elegans protein (17),and human LRP (12, 13), respectively (Fig. 3). These cysteineresidues are likely to be involved in intra- and/or intermo-lecular disulfide bonds necessary for proper tertiary andquaternary structure formation. The differences in the num-bers and sites of such cysteine residues suggest that theseproteins may have different complex structures. Rat gp330/megalin and RAP associate in the endoplasmic reticulum toform large multimeric heterooligomers (7-9). The distributionpatterns and odd numbers of the cysteine residues of theYWTD spacer regions in the LDLR, C. elegans protein, andLRP suggest that some of the cysteines may also be involvedin formation of intermolecular disulfide bonds to form oligo-mers.The similarity of the overall structure between rat gp330/
megalin and the C. elegans protein indicates that the latter ismore related to gp330/megalin than to LRP, although thesethree proteins show comparable amino acid sequence iden-tities in their extracellular regions where the repeat motifarrangements are conserved (Fig. 3). Their significant struc-tural similarity suggests that the C. elegans protein may be ahomologue ofgp330/megalin rather than representing a com-mon ancestral form of gp330/megalin and LRP. However, itis also possible that the C. elegans protein may be a distinctmember of the LDLR gene family. Conservation of theprotein structure over evolution from nematodes to mammalsindicates that gp330/mepalin should play an important bio-logical role.The physiological function of gp330/megalin remains un-
clear, although its localization in coated pits at the cellsurface (1-3) and its multiple ligand-binding domain structurecharacteristic of the LDLR gene family (11, 12) and ligand-binding studies (18, 20, 23) collectively support the notionthat it is an endocytic receptor for multiple ligands. ]Further-more, its appearance in the early embryo (35) and its expres-sion in F9 embryonal carcinoma cells (R.-P. Czek y, R. A.Orlando, and M.G.F., unpublished data) suggest that it is
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required for embryonic development. Interestingly, gp330/megalin has also been shown to bind Ca2+ in the kidneyproximal tubule (23). In addition, monoclonal antibodies thatrecognize a calcium sensing protein in human parathyroidcells have been shown to recognize a 500-kDa protein and tostain the brush border of the kidney proximal tubule andcytotrophoblast cells of the placenta (36, 37), suggesting thatthe parathyroid calcium sensing protein may correspond togp330/megalin or a very similar protein. The availability ofthe gp330/megalin cDNA will greatly facilitate studies de-signed to clarify the physiological functions ofgp330/megalinand its role in HN.
Note Added In Proof. While this paper was in press, a paper waspublished reporting an 804-nt cDNA sequence encoding part of the500-kDa calcium sensing protein ofhuman parathyroid and placentalcytotrophoblast cells (38). The deduced 268-aa sequence shows 81%indentity with residues 4116-4383 of rat gp300/megalin (Fig. 2),indicating that the calcium sensing protein is very likely to be ahuman homologue of gp330/megalin.
We thank Dr. Russell F. Doolittle (University of California, SanDiego) for his generosity in providing advice and assistance with theamino acid sequence analysis. This work was supported by NationalInstitutes of Health Grant DK17724 to M.G.F.
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