Residues Throughout the Cytoplasmic Domain Affect the Internalization Efficiency of P-selectin

doi:10.1074/jbc.270.45.26818

Residues Throughout the Cytoplasmic Domain Affect the Internalization Efficiency of P-selectin (*)

From the (1) From theW. K. Warren Medical Research Institute, Departments of
(2) Medicine and
(3) Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, the
(4) Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104, and the
(5) Department of Cell Biology, University of Virginia School of Medicine, Charlottesville, Virginia 22908

^§ To whom correspondence should be addressed:
W. K. Warren Medical Research Institute, University of Oklahoma Health Sciences Center, 825 N.E. 13th St., Oklahoma City, OK 73104
. Tel.: 405-271-6480; Fax: 405-271-3137.

Abstract

The cytoplasmic domains of many membrane proteins have short sequences, usually including a tyrosine or a di-leucine, that function as sorting signals. P-selectin is an adhesion receptor for leukocytes that is expressed on activated platelets and endothelial cells. Its 35-residue cytoplasmic domain contains signals for sorting into regulated secretory granules, for endocytosis, and for movement from endosomes to lysosomes. The domain has a membrane-distal sequence, YGVFTNAAF, that resembles some tyrosine-based signals. We studied the effects of deletions and mutations in the cytoplasmic tail of human P-selectin on its internalization in clathrin-coated pits of transfected Chinese hamster ovary cells. Mutations and deletions in the putative tyrosine-based motif did not clearly implicate these residues as critical components of a short internalization signal. Indeed, a construct containing a truncated 18-residue cytoplasmic domain with a single substitution (K761A/H773Stop) was internalized nearly three times as fast as wild-type P-selectin; this construct contained no di-leucine, tyrosine, or other known sorting motif. Substitution of residues throughout the cytoplasmic domain affected the internalization rate of P-selectin. Furthermore, the cytoplasmic domain of P-selectin mediated faster internalization when attached to the extracellular and transmembrane domains of the low density lipoprotein receptor than when attached to the corresponding domains of P-selectin. Thus, we were unable to identify a short internalization signal in the cytoplasmic tail of P-selectin. Residues throughout the cytoplasmic domain, and perhaps the transmembrane sequence to which the domain is attached, affect the efficiency of internalization.

Footnotes

↵* This work was supported by Grant HL 34363 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

CHO

Chinese hamster ovary

BSA

bovine serum albumin

FBS

fetal bovine serum

HBSS

Hanks' balanced salt solution

LDL

low density lipoprotein

mAb

monoclonal antibody

NHS

N-hydroxysuccinimide

TBS

Tris-buffered saline

MESNA

2-mercaptoethanesulfonate

ELISA

enzyme-linked immunosorbent assay.
- Received August 3, 1995.
- Revision received September 12, 1995.