Gonadotropin-releasing Hormone Receptor Microaggregation

doi:10.1074/jbc.M007850200

Gonadotropin-releasing Hormone Receptor Microaggregation

RATE MONITORED BY FLUORESCENCE RESONANCE ENERGY TRANSFER*

From the Oregon Regional Primate Research Center and Department of Physiology and Pharmacology, Oregon Health Sciences University, Beaverton, Oregon 97006

Abstract

Gonadotropin-releasing hormone (GnRH) regulates pituitary gonadotropin release and is a therapeutic target for human and animal reproductive diseases. In the present study we have utilized the technique of fluorescence resonance energy transfer to monitor the rate of GnRH receptor-receptor interactions. This technique relies on the observation that the degree of physical intimacy of molecules can be assessed by the tendency of proximal fluorophores to exchange energy. Our data indicate that GnRH agonist, but not antagonist, occupancy of the GnRH receptor promotes physical intimacy (microaggregation) between receptors. The time course indicates that this occurs promptly (<1 min) after occupancy and persists for at least 80 min and within the physiologically relevant range of the releasing hormone. The process measured is not inhibited by 0.1 mm vinblastin, 2 μm cytochalasin D, or 3 mm EGTA, an observation that distinguishes it frommacroaggregation (patching, capping, and internalization). These observations, along with reports from other laboratories, are consonant with a growing body of evidence that indicates that microaggregation is an early event following agonist occupancy of the receptor and part of the mechanism by which effector regulation occurs.

Footnotes

↵* This work was supported by Grants HD19899, HD18185, and RR00163 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 and requests for reprints should be addressed: 505 NW 185th Ave., Beaverton, OR 97006. Tel.: 503-690-5297; Fax: 503-690-5569; E-mail: connm@ohsu.edu.
Published, JBC Papers in Press, October 16, 2000, DOI 10.1074/jbc.M007850200
Abbreviations:

GnRH

gonadotropin-releasing hormone

GPCR

G-protein-coupled receptor

FRET

fluorescence resonance energy transfer

GnRHR

GnRH receptor

GFP

green fluorescent protein

DMEM

Dulbecco's modified Eagle's medium

PCR

polymerase chain reaction

RFP

red fluorescent protein

Nal

2-naphthylalanine

Nal-Arg

Ac-d-Nal¹-d-4-chlorophenylalanine²-d-3-pyridylalanine³, Arg⁵-d-Arg⁶-d-Ala¹⁰-GnRH

rGnRHR

rat GnRHR

FCS

fetal calf serum
- Received August 28, 2000.
- Revision received October 3, 2000.
The American Society for Biochemistry and Molecular Biology, Inc.