Functional Characterization of the LR11 Cytoplasmic Domain
Tanya Williams, Katrin Offe, Guofu Fang, James J. Lah, and Allan I. Levey
Center for Neurodegenerative Disease, Emory University, Atlanta, Georgia

Abstract

LR11 is a recently identified member of the low-density-lipoprotein receptor family. Prior studies in our lab indicate that reduced LR11 expression levels correlate with Alzheimer’s Disease pathology. To elucidate the mechanism underlying this correlation we must first characterize the structural and functional relationships of the LR11 protein. Here we analyze the role of the LR11 cytoplasmic domain in protein trafficking. We posit that deletion of the cytoplasmic domain which contains an internalization motif will impair normal LR11 protein internalization and trafficking. To test our hypothesis we first generated a truncated construct of the LR11 protein that lacks the cytoplasmic domain via cloning approaches. We then verified expression of the deletion construct in comparison to wildtype LR11 protein through Western blotting techniques showing that truncated LR11 protein expresses at levels equivalent to wildtype protein. Lastly we evaluated the cellular localization of the truncated LR11 protein using double-labeling fluorescence immunocytochemistry. Whereas wildtype LR11 protein localizes predominantly to small punctate endosomal compartments within the cell truncated LR11 protein appears to localize closer to the plasma membrane. We will quantify and statistically analyze the observed changes in intracellular localization patterns between the truncated and wildtype protein. Overall our studies demonstrate the promising nature of our chosen approach to LR11 domain analysis and the importance of the cytoplasmic domain to normal LR11 trafficking.

Introduction

Lipid metabolism plays an important but as yet poorly understood role in the pathogenesis of Alzheimer’s Disease (AD). Apolipoprotein E (ApoE) is the principal cholesterol carrier protein in the brain and an ApoE polymorphism e4 is the major genetic risk factor for AD. ApoE interacts with and accelerates accumulation of amyloid b-protein (Ab) the primary component of the extracellular plaque deposits that characterize all forms of AD. How ApoE modulates Ab deposition is unknown. LR11 is a novel ApoE receptor and a member of the low-density lipoprotein receptor (LDL-R) family known to internalize extracellular ligands for lysosomal degradation. Other LDL-R family members have been implicated in Ab production or clearance. Our laboratory identified LR11 as a novel reduced transcript and protein in AD as compared to age-matched controls and has recently demonstrated that LR11 overexpression reduces extracellular Ab. Hence the relationship between LR11 biology and AD pathology merits further investigation. Here we analyze the role of the cytoplasmic domain in LR11 protein trafficking. We hypothesize that deletion of the cytoplasmic domain will impair LR11 internalization and trafficking. To confirm our hypothesis we will generate a truncated LR11 construct and access function via immunoblotting and immunocytochemical approaches.

Methods and Materials

cDNA constructs

To construct the LR11 cytoplasmic domain deletion (LR11-∆) plasmid truncated LR11 cDNA was amplified from the pSorLA/cDNA3 vector by PCR with a 5' primer (5'-AGTCCAGAATTCAACATGGCGACACGGAGC-3') and a 3' primer (5'-ATCTCACTCGAGTTATCTGGCTGCCTGCGT-3'). The products were digested and ligated into the pcDNA 3.1 zeo (+) vector. Competent cells were transformed exposed to ampicillin and growing clones were selected and purified. Purified clones were re-digested to verify correct LR11-∆ insertion.

Cell culture and transfections

Human embryonic kidney (HEK 293) cells were maintained at 37ºC and 5% CO2 in DMEM (Mediatech) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin-streptomycin. Cells were transfected with LR11 or pcDNA plasmids using FuGENE 6 (Roche). Immunocytochemistry was performed 2 days after transfection.

Immunoblotting

For immunoblot analyses total cell extracts were prepared by harvesting cells in phosphate buffered saline with protease inhibitors (CompleteTM; Boehringer-Mannheim). Cells were pelleted and extracted on ice for 60 min in IP buffer. Cell extract and conditioned medium samples were separated across a 7.5% SDS-PAGE gel and transferred overnight to Immobilon-P transfer membranes (Millipore). Blots were blocked in tris-buffered saline with blocking buffer at room temperature and then probed with LR11 antibodies overnight at 4ºC. Blots were washed and incubated with a fluorophore conjugated secondary antibody for 1 hr at room temperature. Blots were visualized using a LI-COR Odyssey Infrared Imager.

Immunocytochemistry

Cells were fixed for 30 min in 2% paraformaldehyde then blocked and permeabilized for 30 min. Cells were incubated overnight at 4ºC with LR11 and organelle specific marker primary antibodies. For double labeling primaries were incubated together. The cells were rinsed and incubated with rhodamine- or CY5-conjugated secondary antibodies (Jackson Immunoresearch). For double labeling secondary antibodies were incubated together. Control incubations included omission of primary antibodies to test nonspecific secondary antibody binding. Cells were scanned using a Zeiss (Heidelberg) LSM 510 laser scanning confocal microscope.

Results

LR11 Antibodies Recognize Different Protein Domains. Full length LR11 protein consists of several extracellular domains a transmembrane domain and a cytoplasmic domain. As our studies aim to characterize LR11 cytoplasmic domain function we created a truncated LR11 construct as pictured in the schematic diagram on the left

(Figure 1).

While full length LR11 protein has epitopes for both extracellular and cytoplasmic antibodies the deletion construct lacks the epitope for the cytoplasmic antibody. Therefore the cytoplasmic antibody is specific for full length LR11 protein only and can be used to differentiate between the two LR11 constructs in later assays. LR11-D Protein Expresses at Levels Equivalent to LR11wt. The cell extract immunoblot above

(Figure 2)

shows that LR11-D protein is detectably shorter than LR11wt protein and expresses at equivalent levels. Thus we successfully created a truncated construct that can be used in further assays. Additionally immunocytochemical

(Figure 3)

and immunoblot results confirm that the cytoplasmic domain antibody only recognizes LR11wt protein while extracellular antibodies recognize LR11wt and LR11-D protein. LR11-D Protein Exhibits Reduced Localization to Early Endosomes. Unlike LR11wt protein which localizes predominantly to early endosome compartments and vesicles as shown in the top 3 panels

(Figure 4)

LR11-D protein (visualized using an antibody directed to the extracellular domain) departs from wildtype intracellular localization patterns and exhibits reduced overlap with the endosome specific marker Early Endosome Antigen 1 (EEA1). LR11-D Protein Displays Increased Localization to the Plasma Membrane. Using double-labeling fluorescence immunocytochemistry LR11-D protein appears to localize closer to the plasma membrane as increased overlap between the plasma membrane marker Na+/K+ATPase and LR11-D protein is observed in the bottom panels

(Figure 5).

LR11wt protein again displays localization to punctate vesicles. LR11-D Conditioned Media Shows Increased LR11 Protein Levels. Unexpectedly conditioned media collected from cells transfected with the LR11-D construct and probed with an extracellular LR11 antibody show increased LR11 protein levels as compared to media from vector or LR11wt transfected cells

(Figure 6).

Conclusions and Future Studies

We were able to successfully generate a truncated LR11 construct for use in subsequent assays. LR11-D protein is smaller than LR11wt protein and expresses at similar levels. LR11wt protein localizes to small punctate endosomal compartments within the cell. LR11-D protein does not follow wildtype localization patterns but localizes closer to the plasma membrane. Therefore the LR11 cytoplasmic domain is important for normal trafficking patterns. LR11-D conditioned media shows higher levels of LR11 protein than wildtype or control which may indicate that impaired receptor trafficking increases the rate of LR11 ectodomain shedding at the plasma membrane. Future directions include quantitative analysis of the observed colocalization results and the creation of additional constructs deleting restricted portions of the cytoplasmic domain to further characterize its role in LR11 protein trafficking.

Acknowledgements and Funding Attributions

The authors would like to thank members of the Levey laboratory for technical and intellectual support. This material is based upon work supported by the SURE Program and the Howard Hughes Medical Institute under Grant No.52003727. References: 1) Puglielli L Tanzi R. and Kovacs D. (2003) Alzheimer’s disease: the cholesterol connection. Nature Neuroscience 6 (4): 345-351. 2) Van Uden E Mallory M Veinbergs I Alford M Rockenstein E and Masliah E. (2002) Increased extracellular amyloid deposition and neurodegeneration in human amyloid precursor protein transgenic mice deficient in receptor-associated protein. The Journal of Neuroscience 22(21): 9298-9304. 3) Scherzer CR Gearing M Heilman C Gutekunst C Fang G Lah J Wainer B and Levey AI. Profiling of Alzheimer’s lymphoblasts: apoE receptor LR11 expression mirrors changes in the brain. Submitted. 4) Hampe W Urny J Franke I Hoffmeister-Ullerich SAH Herrmann D Petersen CM Lohmann J and Schaller HC. (1999) A head-activator binding protein is present in hydra in a soluble and a membrane anchored form. Development 126: 4077-4086.

In Plain English

LR11 is a recently identified member of the low-density-lipoprotein receptor family. Prior studies in our lab indicate that brain tissue from Alzheimer's Disease patients show reduced levels of LR11 protein in comparison to age-matched contorls. To understand the correlation between LR11 protein levels and Alzheimer's Disease we must first characterize the structural and functional relationships of the LR11 protein. Here we analyze the role of the LR11 cytoplasmic domain in protein trafficking. We posit that deletion of the cytoplasmic domain will alter normal LR11 receptor trafficking from the cell membrane to internal compartments. To test our hypothesis we first deleted the cytoplasmic domain via cloning approaches. We show that truncated LR11 protein expresses at levels equivalent to full length protein. We also indicate that full length LR11 protein is found primarily in small endosomal compartments within the cell while truncated LR11 protein appears to localize closer to the plasma membrane. We will quantify and statistically analyze the observed changes in intracellular localization patterns between the truncated and full length protein. Overall our studies demonstrate the promising nature of our chosen approach to LR11 domain analysis and the importance of the cytoplasmic domain to normal LR11 trafficking.