Kalirin, a Multifunctional PAM COOH-terminal Domain Interactor Protein, Affects Cytoskeletal Organization and ACTH Secretion from AtT-20 Cells

doi:10.1074/jbc.274.5.2929

Kalirin, a Multifunctional PAM COOH-terminal Domain Interactor Protein, Affects Cytoskeletal Organization and ACTH Secretion from AtT-20 Cells*

From the Departments of Neuroscience and Physiology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, the^‡Departments of Surgery and Physiology, The University of Maryland Medical School, Baltimore, Maryland 21201, and the ^§Institute of Biomedicine, Department of Anatomy, University of Helsinki, Helsinki, Finland 00014

Abstract

The production and regulated secretion of bioactive peptides require a series of lumenal enzymes to convert inactive precursors into bioactive peptides plus several cytosolic proteins to govern granule formation, maturation, translocation, and exocytosis. Peptidylglycine α-amidating monooxygenase (PAM), an enzyme essential for biosynthesis of many peptides, is an integral membrane protein with trafficking information in both its lumenal and cytosolic domains. Kalirin, a PAM cytosolic domain interactor protein with spectrin-like repeats and GDP/GTP exchange factor activity for Rac1, is expressed with PAM in neurons but is not expressed in the anterior pituitary or AtT-20 corticotrope cells. Expression of Kalirin alters the cytoskeletal organization of Chinese hamster ovary and AtT-20 cells expressing membrane PAM. Expression of membrane PAM also alters cytoskeletal organization, demonstrating the presence of endogenous proteins that can mediate this effect. Significant amounts of both PAM and Kalirin fractionate with cytoskeletal elements. Since cytoskeletal organization is critical for exocytosis, constitutive-like and regulated secretions were evaluated. Whereas the constitutive-like secretion of adrenocorticotropic hormone (ACTH) is increased by expression of membrane PAM, regulated secretion is eliminated. Expression of Kalirin in AtT-20 cells expressing membrane PAM restores stimulated secretion of ACTH. Thus, Kalirin or its homologue may be essential for regulated secretion, and the PAM-Kalirin interaction may coordinate intragranular with cytosolic events.

Footnotes

↵* This work was supported by Grant DK32948 from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¶ To whom correspondence should be addressed: The Johns Hopkins University School of Medicine, 725 N. Wolfe St., Baltimore, MD 21205. Tel.: 410-955-6937; Fax: 410-955-0681; E-mail: beipper{at}jhmi.edu.
Abbreviations:

PAM

peptidylglycine α-amidating monooxygenase

ACTH

adrenocorticotropic hormone

CHO

Chinese hamster ovary

nt

nucleotide

DH

Dbl homology

POMC

proopiomelanocortin

FITC

fluorescein isothiocyanate

Ab

antibody.
- Received June 11, 1998.
- Revision received November 19, 1998.
The American Society for Biochemistry and Molecular Biology, Inc.