Agonist-regulated Interaction between α2-Adrenergic Receptors and Spinophilin

Jeremy G Richman,Ashley E Brady,Qin Wang,Jennifer L Hensel,Roger J Colbran,Lee E Limbird

doi:10.1074/jbc.m011679200

Jeremy G Richman, Ashley E Brady + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m011679200

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Abstract

Previously, we demonstrated that the third intracellular (3i) loop of the heptahelical alpha2A-adrenergic receptor (alpha2A AR) is critical for retention at the basolateral surface of polarized Madin-Darby canine kidney II (MDCKII) cells following their direct targeting to this surface. Findings that the 3i loops of the D2 dopamine receptors interact with spinophilin (Smith, F. D., Oxford, G. S., and Milgram, S. L. (1999) J. Biol. Chem. 274, 19894-19900) and that spinophilin is enriched beneath the basolateral surface of polarized MDCK cells prompted us to assess whether alpha(2)AR subtypes might also interact with spinophilin. [35S]Met-labeled 3i loops of the alpha2A AR (Val(217)-Ala(377)), alpha2BAR (Lys(210)-Trp(354)), and alpha2CAR (Arg(248)-Val(363)) subtypes interacted with glutathione S-transferase-spinophilin fusion proteins. These interactions could be refined to spinophilin amino acid residues 169-255, in a region between spinophilin's F-actin binding and phosphatase 1 regulatory domains. Furthermore, these interactions occur in intact cells in an agonist-regulated fashion, because alpha2A AR and spinophilin coimmunoprecipitation from cells is enhanced by prior treatment with agonist. These findings suggest that spinophilin may contribute not only to alpha2 AR localization but also to agonist modulation of alpha2AR signaling.

Highlights

The three ␣2-adrenergic receptor (␣2AR)1 subtypes are members of the type II, biogenic amine-binding, G protein-coupled receptor family
We demonstrated that the third intracellular (3i) loop of the heptahelical ␣2A-adrenergic receptor (␣2AAR) is critical for retention at the basolateral surface of polarized Madin-Darby canine kidney II (MDCKII) cells following their direct targeting to this surface
Chem. 274, 19894 –19900) and that spinophilin is enriched beneath the basolateral surface of polarized MDCK cells prompted us to assess whether ␣2AR subtypes might interact with spinophilin. [35S]Met-labeled 3i loops of the ␣2AAR (Val217-Ala377), ␣2BAR (Lys210-Trp354), and ␣2CAR (Arg248-Val363) subtypes interacted with glutathione S-transferase-spinophilin fusion proteins

Summary

EXPERIMENTAL PROCEDURES

Materials—The pGEMEX-2 vector and TnT in vitro translation kit were from Promega (Madison, WI). Horseradish peroxidase-labeled anti-mouse and anti-rat antibodies were from Amersham Pharmacia Biotech. MDCKII Cell Culture and Polarization—MDCKII cells were plated at confluence (ϳ1–2.5 ϫ 105 cells) and grown on 12-mm Transwell filters (0.4-␮m pore size, Costar, Cambridge, MA) in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (Sigma) and 100 units/ml penicillin and 10 ␮g/ml streptomycin at 37 °C/5% CO2 as described previously [19] except with daily media changes for 5–7 days. Under these conditions, cells form a monolayer and functionally polarize with distinct apical and basolateral surfaces separated by tight junctions. Leaks range from 5–10%, and we discard from study any culture wells of Ͼ10% leak

Immunofluorescent Labeling and Confocal Microscopy

Western Blot Analysis

RESULTS

DISCUSSION