Involvement of a Heterotrimeric G Protein α Subunit in Tight Junction Biogenesis

doi:10.1074/jbc.271.42.25750

Involvement of a Heterotrimeric G Protein α Subunit in Tight Junction Biogenesis*

From the Renal Division, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115

^‡ To whom correspondence should be addressed:
Renal Division, Thorn 628, Brigham and Women's Hospital, 75 Francis St., Boston, MA 02115.
Tel.: 617-732-5809; Fax: 617-732-6392; E-mail: denker{at}bustoff.bwh.harvard.edu

Abstract

The tight junction (TJ) of polarized epithelial cells is critical for maintaining an impermeant barrier and epithelial cell polarity. The signaling events important for TJ assembly require regulated calcium stores and protein kinase C (PKC), but the earliest signaling events in the cascade have not been well defined. We now show that Gα_i2 in Madin Darby canine kidney (MDCK) cells localizes to a region overlapping with the TJ. To further analyze the localization of Gα subunits in epithelial cells, rat Gα_o, Q205Lα_o (Gα_o “activated” by point mutation) and plasmid without insert (PC) were transfected into MDCK cells and localized by immunofluorescence and confocal microscopy. Similar to endogenous Gα_i2, Gα_o-MDCK cells localize Gα_o, (84% similar to Gα_i2) in the subapical region overlapping with ZO-1 (zona occludens-I), a key component of the TJ. PC-MDCK cells have no detectable Gα_o. In Gα_o-MDCK cells, a physical association of Gα_o with components of the TJ was detectable by immunoprecipitation of ZO-1. Immunoprecipitates of ZO-1 from Gα_o-MDCK cells consistently coprecipitated Gα_o. Constitutively active Q205LGα_o localized to the subapical lateral membrane similar to wild-type Gα_o. To determine if constitutively activated Gα subunits can affect TJ biogenesis, the formation of tight junctions in PC, Gα_o, and Q205Lα_o-MDCK cells was followed by measurement of transepithelial resistance (TER) during the Ca²⁺ switch, a model widely used to study mechanisms of junctional assembly. Baseline and post Ca²⁺ switch TER values did not differ among the cell lines. However, constitutively activated Q205Lα_o-MDCK cells developed TER significantly faster than PC and Gα_o cells in the early phase (0-4 h) (54 ± 4 versus 23 ± 3 (PC); 12 ± 1 (Gα_o) Ω·m²/h) and late phase (4-h peak) (117 ± 10 versus 45 ± 5 (PC); 66 ± 7 (Gα_o) Ω·cm²/h) after Ca²⁺ switch. Peak TER values were significantly higher in Q205Lα_o-MDCK cells (1168 ± 107 versus 437 ± 37 (PC); 548 ± 54 (Gα_o) Ω·cm²). These results indicate that Gα_o and Q205Lα_o expressed in MDCK cells are localized near the junctional complex, associate with at least one TJ protein, and that activated Gα_o accelerates TJ biogenesis without significantly affecting the maintenance of the TJ. Together, these results suggest an important role for heterotrimeric G proteins in TJ assembly.

Footnotes

↵^§ Established Investigator of the American Heart Association. Supported by an RO1 grant from NIDDK, National Institutes of Health.
↵* This work was supported in part by National Institutes of Health Clinical Investigator Award DK02110, National Kidney Foundation Young Investigator Grant, and American Heart Association (Massachusetts Affiliate) Beginning Grant in Aid (to B. M. D.). 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.
↵¹ The abbreviations used are:

TJ

tight junction

G protein

guanine nucleotide-binding protein

ZO-1

zona occludens-1

TER

transepithelial resistance

MDCK

Madin Darby canine kidney

PKC

protein kinase C

LC

low calcium

NC

normal calcium

PAGE

polyacrylamide gel electrophoresis

PBS

phosphate-buffered saline.
- Received July 18, 1996.
- Revision received August 19, 1996.