Regulation of Constitutive Exocytic Transport by Membrane Receptors

doi:10.1074/jbc.271.7.3523

Regulation of Constitutive Exocytic Transport by Membrane Receptors

A BIOCHEMICAL AND MORPHOMETRIC STUDY (*)

From the Istituto di Ricerche Farmacologiche Mario Negri, Consorzio Mario Negri Sud, Laboratory of Molecular Neurobiology and the
Physiopathology of Secretion Unit, 66030 S. Maria Imbaro (Chieti), Italy

^§To whom correspondence should be addressed:
Laboratory of Molecular Neurobiology, Consorzio Mario Negri Sud, 66030 S. Maria Imbaro (Chieti), Italy.
Tel.: 39-872-570354; Fax: 39-872-578240.

↵** Present address: Depts. of Cell and Molecular Biology, The Scripps Research Inst., La Jolla, CA 92037.
↵^§§ Present address: Laboratorio di Biologia Cellulare e dello Sviluppo, Università di Pisa, Via Carducci 13, 56010 Ghezzano (PI) Italy.

Abstract

Biochemical and morphometric approaches were combined to examine whether constitutive secretory transport might be controlled by plasma membrane receptors, as this possibility would have significant physiological implications. Indeed, IgE receptor stimulation in rat basophilic leukemia cells potently increased the rate of transport of soluble pulse-labeled S-sulfated glycosaminoglycans from distal Golgi compartments to the cell surface. This effect was largely protein kinase C (PKC)-dependent. Direct activation of PKC also stimulated constitutive transport of glycosaminoglycans, as indicated by the use of agonistic and antagonistic PKC ligands. PKC ligands also had potent, but different, effects on the exocytic transport from distal Golgi compartments to the plasma membrane of a membrane-bound protein (vesicular stomatitis virus glycoprotein), which was slightly stimulated by activators and profoundly suppressed by inhibitors of PKC. Morphological analysis showed impressive changes of the organelles of the secretory pathway in response to IgE receptor stimulation and to direct PKC activation (enhanced number of buds and vesicles originating from the endoplasmic reticulum and Golgi and increase in surface and volume of Golgi compartments), suggestive of an overall activation of exocytic movements. These results show that rapid and large changes in constitutive transport fluxes and in the morphology of the exocytic apparatus can be induced by membrane receptors (as well as by direct PKC stimulation).

Footnotes

↵^¶ Recipients of a postdoctoral fellowship from the Ministero del Lavoro-POFSE 936106 I1 “Esperto in tecnologie farmacologiche e biomediche avanzate nel campo della ricerca farmaceutica e agroalimentare.”
↵* This work was supported in part by the Italian National Research Council (Convenzione CNR-Consorzio Mario Negri Sud). 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:

ER

endoplasmic reticulum

PKC

protein kinase C

TG/TGN

trans Golgi/trans Golgi network

ARF

ADP-ribosylation factor

RBL

rat basophilic leukemia

BFA

brefeldin A

PMA

phorbol 12-myristate 13-acetate

DNP

dinitrophenol

BSA

bovine serum albumin

DPP

12-deoxyphorbol 13-phenylacetate

DPPA

12-deoxyphorbol 13-phenylacetate 20-acetate

VSV

vesicular stomatitis virus

MEM

Eagle's minimal essential medium

DMEM

Dulbecco's modified Eagle's medium

FCS

fetal calf serum

PAGE

polyacrylamide gel electrophoresis

GAG

free glycosaminoglycan chains

VSV-G

VSV glycoprotein

PBS

phosphate-buffered saline

MCV

mean cell volume

MAC

mean area of cell projections

MAC

mean area of cell sections

TVC

tubular-vesicular clusters

DAG

diacylglycerol.
↵²R. Buccione, S. Bannykh, I. Santone, M. Baldassarre, F. Facchiano, Y. Bozzi, G. Di Tullio, A. Mironov, A. Luini, and M. A. De Matteis, unpublished observations.
↵³Combining morphometric and transport data, one might expect, based on the effect on GAG release and on the t of release of GAG (considered as a marker of TG/TGN bulk volume; Fig. 2 and Fig. 3), a decrease in size of the TG/TGN within the first several minutes of PMA treatment. However, while this decrease might in fact occur, it would be undetectable for technical reasons, since in our experiments the tubular structures of the TG/TGN cannot be reliably distinguished from the cis Golgi tubules. Instead, since all Golgi elements are recorded together for quantitative estimations, the possible membrane loss from the TG/TGN is more than counterbalanced by the increased input into the cis Golgi from the ER, whose size is 5-10-fold larger than that of the Golgi complex (Table 1).
↵⁴S. Bannykh and W. Balch, personal communication.
- Received August 3, 1995.
- Revision received October 12, 1995.