Signaling Complexes Associated with the Type I Gonadotropin-Releasing Hormone (GnRH) Receptor: Colocalization of Extracellularly Regulated Kinase 2 and GnRH Receptor within Membrane Rafts

Abstract

Our previous work demonstrated that the type I GnRH receptor (GnRHR) resides exclusively and constitutively within membrane rafts in alphaT3-1 gonadotropes and that this association was necessary for the ability of the receptor to couple to the ERK signaling pathway. G(alphaq), c-raf, and calmodulin have also been shown to reside in this compartment, implicating a raft-associated multiprotein signaling complex as a functional link between the GnRHR and ERK signaling. In the studies reported here, we used subcellular fractionation and coimmunoprecipitation to analyze the behavior of ERKs with respect to this putative signaling platform. ERK 2 associated partially and constitutively with low-density membranes both in alphaT3-1 cells and in whole mouse pituitary. Cholesterol depletion of alphaT3-1 cells reversibly blocked the association of both the GnRHR and ERKs with low-density membranes and uncoupled the ability of GnRH to activate ERK. Analysis of the kinetics of recovery of ERK inducibility after cholesterol normalization supported the conclusion that reestablishment of the association of the GnRHR and ERKs with the membrane raft compartment was not sufficient for reconstitution of signaling activity. In alphaT3-1 cells, the GnRHR and ERK2 coimmunoprecipitated from low-density membrane fractions prepared either in the presence or absence of detergent. The GnRHR also partitioned into low-density, detergent-resistant membrane fractions in mouse pituitary and coimmunoprecipitated with ERK2 from these fractions. Collectively, these data support a model in which coupling of the GnRHR to the ERK pathway in gonadotropes involves the assembly of a multiprotein signaling complex in association with specialized microdomains of the plasma membrane.