Linda Jacobsen

Molecular Identification of a Novel Candidate Sorting Receptor Purified from Human Brain by Receptor-associated Protein Affinity Chromatography

Receptor-associated protein (RAP) is an endoplasmic reticulum/Golgi protein involved in the processing of receptors of the low density lipoprotein receptor family. A ∼95-kDa membrane glycoprotein, designated gp95/sortilin, was purified from human brain extracts by RAP affinity chromatography and cloned in a human cDNA library. The gene maps to chromosome 1p and encodes an 833-amino acid type I receptor containing an N-terminal furin cleavage site immediately preceding the N terminus determined in the purified protein. Gp95/sortilin is expressed in several tissues including brain, spinal cord, and testis. Gp95/sortilin is not related to the low density lipoprotein receptor family but shows intriguing homologies to established sorting receptors: a 140-amino acid lumenal segment of sortilin representing a hitherto unrecognized type of extracellular module shows extensive homology to corresponding segments in each of the two lumenal domains of yeast Vps10p, and the extreme C terminus of the cytoplasmic tail of sortilin contains the casein kinase phosphorylation consensus site and an adjacent dileucine sorting motif that mediate assembly protein-1 binding and lysosomal sorting of the mannose-6-phosphate receptors. Expression of a chimeric receptor containing the cytoplasmic tail of gp95/sortilin demonstrates evidence that the tail conveys colocalization with the cation-independent mannose6-phosphate receptor in endosomes and the Golgi compartment.

Molecular characterization of a novel human hybrid-type receptor that binds the alpha2-macroglobulin receptor-associated protein

The 39-40-kDa receptor-associated protein (RAP) binds to the members of the low density lipoprotein receptor gene family and functions as a specialized endoplasmic reticulum/Golgi chaperone. Using RAP affinity chromatography, we have purified a novel ∼250-kDa brain protein and isolated the corresponding cDNA. The gene, designated SORL1, maps to chromosome 11q 23/24 and encodes a 2214-residue type 1 receptor containing a furin cleavage site immediately preceding the N terminus determined in the purified protein. The receptor, designated sorLA-1, has a short cytoplasmic tail containing a tyrosine-based internalization signal and a large external part containing (from the N-terminal): 1) a segment homologous to domains in the yeast vacuolar protein sorting 10 protein, Vps10p, that binds carboxypeptidase Y, 2) five tandemly arranged YWTD repeats and a cluster of 11 class A repeats characteristic of the low density lipoprotein receptor gene family receptors, and 3) six tandemly arranged fibronectin type III repeats also found in certain neural adhesion proteins. sorLA-1 may therefore be classified as a hybrid receptor. Northern blotting revealed specific mRNA transcripts in brain, spinal cord, and testis but not in several major organs. Both RAP and an antibody against a synthetic peptide derived from a sequence determined in the mature protein detected sorLA-1 in crude human brain extracts. The domain structure suggests that sorLA-1 is an endocytic receptor possibly implicated in the uptake of lipoproteins and of proteases.

Propeptide cleavage conditions sortilin/neurotensin receptor‐3 for ligand binding

We recently reported the isolation and sequencing of sortilin, a new putative sorting receptor that binds receptor‐associated protein (RAP). The luminal N‐terminus of sortilin comprises a consensus sequence for cleavage by furin, R41WRR44, which precedes a truncation originally found in sortilin isolated from human brain. We now show that the truncation results from cellular processing. Sortilin is synthesized as a proform which, in late Golgi compartments, is converted to the mature receptor by furin‐mediated cleavage of a 44 residue N‐terminal propeptide. We further demonstrate that the propeptide exhibits pH‐dependent high affinity binding to fully processed sortilin, that the binding is competed for by RAP and the newly discovered sortilin ligand neurotensin, and that prevention of propeptide cleavage essentially prevents binding of RAP and neurotensin. The findings evidence that the propeptide sterically hinders ligands from gaining access to overlapping binding sites in prosortilin, and that cleavage and release of the propeptide preconditions sortilin for full functional activity. Although proteolytic processing is involved in the maturation of several receptors, the described exposure of previously concealed ligand‐binding sites after furin‐mediated cleavage of propeptide represents a novel mechanism in receptor activation.

Crystallization and preliminary X-ray analysis of methylamine-treated α2-macroglobulin and 3 α2-macroglobulin-proteinase complexes

Crystals of methylamine‐treated α2‐macroglobulin (α2M‐MA), α2‐macroglobulin in complex with two molecules of trypsin, α2M‐T2, one molecule of plasmin, α2‐M‐PL, and one molecule of plasmin followed by methylamine‐treatment, α2M‐PL(MA), have reproducibly been obtained using ammonium sulfate or magnesium sulfate as precipitants. The crystals are fragile tetragonal bipyramids of up to 1.5 mm in length. Crystals of α2M‐MA diffracted to at least 9A˚resolution, crystals of α2M‐T2 diffracted to 10A˚resolution and crystals of α2M‐PL and α2M‐PL(MA) diffracted to 11A˚resolution. For α2M‐MA the cell parameters were determined as:a=b=257A˚,c=555A˚; and for α2M‐T2 as:a=b=247A˚,c=559A˚. For both preparations the space group was I4(1)22. As estimated from density measurements, the crystals of α2M‐MA and α2M‐T2 contain one 360 kDa α2M dimer per asymmetric unit. The volume of the asymmetric unit/molecular weight,V m, was estimated at 5.6A˚3/Da. The crystal parameters of α2M‐PL and α2M‐PL(MA) were not determined.

Crystallisation and preliminary X-ray analysis of the receptor-binding domain of human and bovine α2-macroglobulin

The receptor‐binding domains (RBDs) of human and bovine α 2‐macroglobulin (α 2M) have been isolated after limited proteolysis of methylamine‐treated α 2M with papain. Single crystals of the RBDs have been grown by vapour diffusion. Crystals of human RBD are very thin plates unsuited for data collection. However, crystals of RBD from bovine α 2M give diffraction patterns suitable for X‐ray analysis, and a complete dataset with a maximum resolution of 2.3 Å has been collected with synchrotron radiation at cryogenic temperature. The crystals belong to spacegroup P3121 or P3221 with cell parameters , .

Low-resolution X-ray diffraction data obtained from hexagonal crystals of methylamine-treated alpha2-macroglobulin.

Two hexagonal crystal forms of tetrameric human methylamine-treated alpha(2)-macroglobulin have been grown by vapour diffusion. One of the crystal forms diffracts X-rays beyond 9 A resolution. The space group of this form is P6(2)22 or P6(4)22 with a = b = 327, c = 219 A and with one dimer of alpha(2)-macroglobulin in the asymmetric unit. Several data sets have been collected by the use of synchrotron radiation at cryogenic temperature. A native data set extending to 10 A resolution has been obtained. The merging R factor of these data is 10.3%.