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J. Biol. Chem., Vol. 282, Issue 28, 20502-20512, July 13, 2007

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Effects of pH and Low Density Lipoprotein (LDL) on PCSK9-dependent LDL Receptor Regulation^*

Timothy S. Fisher, Paola Lo Surdo, Shilpa Pandit, Marco Mattu, Joseph C. Santoro, Doug Wisniewski^¶, Richard T. Cummings, Alessandra Calzetta, Rose M. Cubbon, Paul A. Fischer^||, Anil Tarachandani^||, Raffaele De Francesco, Samuel D. Wright, Carl P. Sparrow, Andrea Carfi¹, and Ayesha Sitlani²

From the Departments of Cardiovascular Diseases, ^¶Infectious Diseases, and ^||Target Validation, Merck Research Laboratories, Rahway, New Jersey 07065 and the Istituto di Ricerche di Biologia Molecolare "P. Angeletti", 00040 Pomezia, Rome, Italy

Mutations within PCSK9 (proprotein convertase subtilisin/kexin type 9) are associated with dominant forms of familial hyper- and hypocholesterolemia. Although PCSK9 controls low density lipoprotein (LDL) receptor (LDLR) levels post-transcriptionally, several questions concerning its mode of action remain unanswered. We show that purified PCSK9 protein added to the medium of human endothelial kidney 293, HepG2, and Chinese hamster ovary cell lines decreases cellular LDL uptake in a dose-dependent manner. Using this cell-based assay of PCSK9 activity, we found that the relative potencies of several PCSK9 missense mutants (S127R and D374Y, associated with hypercholesterolemia, and R46L, associated with hypocholesterolemia) correlate with LDL cholesterol levels in humans carrying such mutations. Notably, we found that in vitro wild-type PCSK9 binds LDLR with an 150-fold higher affinity at an acidic endosomal pH (K_D = 4.19 nM) compared with a neutral pH (K_D = 628 nM). We also demonstrate that wild-type PCSK9 and mutants S127R and R46L are internalized by cells to similar levels, whereas D374Y is more efficiently internalized, consistent with their affinities for LDLR at neutral pH. Finally, we show that LDL diminishes PCSK9 binding to LDLR in vitro and partially inhibits the effects of secreted PCSK9 on LDLR degradation in cell culture. Together, the results of our biochemical and cell-based experiments suggest a model in which secreted PCSK9 binds to LDLR and directs the trafficking of LDLR to the lysosomes for degradation.

Received for publication, February 23, 2007 , and in revised form, May 10, 2007.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. I-III.

¹ To whom correspondence may be addressed. Tel.: 39-06-9109-3550; Fax: 39-06-9109-3225; E-mail: andrea_carfi{at}merck.com. ² To whom correspondence may be addressed: Dept. of Cardiovascular Diseases, Merck Research Laboratories, PO Box 2000, Mail Code RY80Y-300, Rahway, NJ 07065. Tel.: 732-594-0788; Fax: 732-594-1169; E-mail: ayesha_sitlani{at}merck.com.

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