Expression of the Gβ5/R7-RGS protein complex in pituitary and pancreatic islet cells

Introduction

Heterotrimeric G protein subunit Gβ₅ is expressed primarily in the nervous system and retina and, among Gβ isoforms, is unique in its ability to heterodimerize with regulator of G protein signaling (RGS) proteins of the R7 subfamily (R7-RGS) [see [1, 2] for recent reviews]. The R7-RGS subfamily consists of RGS proteins 6, 7, 9, and 11 which can form tight heterodimers with the G protein β5 subunit through a Gγ-like (GGL) domain [1, 2]. R7-RGS subfamily members are unstable in the absence of Gβ₅ [3]. Previous study from our laboratory showed that in addition to its expression in brain and neural cell lines, Gβ₅ was also expressed in the αT3-1 gonadotrophic pituitary cell line [4]. We show now that the Gβ₅/R7-RGS complex is expressed in corticotroph-derived pituitary AtT-20 cells, pituitary gland and purified pancreatic islets. The expression of Gβ₅ in various endocrine cell lines and native tissues raises the possibility that hormone secretion, and not just neurotransmission and phototransduction, may be regulated by the Gβ₅/R7-RGS complex.

Materials and methods

Antibodies employed in immunoblots and immunohistochemistry included normal rabbit IgG, rabbit anti-Gβ₅ polyclonal N-terminal antibody ATDG [5], anti-β-actin mouse monoclonal (Sigma, A-5315), mouse anti-α-tubulin (Calibiochem, CP06), mouse anti-ACTH monoclonal antibody (Abcam, ab8615), mouse anti-p84/N5 [5E10] monoclonal antibody (GeneTex, GTX70220), rabbit anti-RGS7 polyclonal antibody 7RC-1 (cross-reacts with RGS6) [6], and rabbit anti-RGS9-2 polyclonal antibody RGS9CT (raised against the synthetic peptide 31-mer H-CRSPRK PFASPSRFIRRPSIAICPSPSRVAL-NH2). Secondary antibodies included HRP-conjugated goat anti-mouse (no. 1858413) and anti-rabbit (no. 1858415) antibodies from Pierce. AtT-20 cells were grown in 75-cm² flasks at 37°C and 5% CO₂ containing DMEM supplemented with 10% bovine serum, 5% horse serum, 4 mM L-glutamine, and penicillin/streptomycin (Biofluids, Rockville, MD). MIN6 cells were cultured in DMEM medium containing 4.5 g/L glucose, 10% fetal bovine serum and penicillin/streptomycin. Fractions of cultured cells were prepared using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce, 78833) according to the manufacturer’s instructions. For isolation of pancreatic islets the procedure of Saeki et al. [7] was followed with minor modification. For immunohistochemistry and immunofluorescence, pituitary glands were excised from the ventral portion of mouse brain and fixed overnight in 4% paraformaldehyde, and then incubated in 70% ethanol. The preserved pituitary was processed for paraffin embedding and microscopic sectioning (Histoserv, Inc., Germantown, MD). Slides were prepared for immunohistochemistry according to the manufacturer’s recommendations (Bethyl Labs, Immunohistochemistry Accessory Kit, no. IHC-101). Confocal laser microscopy employed a Leica SP2-UV 405 confocal microscope (Leica Microsystems, Exton, PA, USA). Mice with a heterozygous deletion of exon 3 of Gnb5 in the germline were kindly provided by Dr. Ching-Kang Jason Chen (Virginia Commonwealth University Medical Center, Richmond, VA). Gnb5 KO pups were generated from heterozygotes as previously described [8].

Results

We looked for expression of Gβ₅ and R7-RGS protein in AtT-20 cells, a well-characterized mouse pituitary tumor-derived cell line that expresses adrenocorticotropic hormone (ACTH). Gβ₅ and RGS7 were expressed in whole cell lysates of AtT-20 cells comparable to their expression in neuron-like PC12 cells (Fig. 1a). Subcellular fractionation showed that in AtT-20 cells, as in mouse brain and PC12 cells [9], Gβ₅ and RGS7 are targeted to both the cytoplasm and the nucleus (Fig. 1b).

Fig. 1.

Expression of Gβ₅ in AtT-20 cells, mouse pituitary, MIN6 insulinoma cells, and purified mouse pancreatic islets. a Whole cell lysates from AtT-20 and PC12 cells were prepared and analyzed for Gβ₅ and RGS7 by SDS-PAGE followed by immunoblotting. The corresponding β-actin levels are shown as loading controls. b Subcellular distribution of Gβ₅ and RGS7 in AtT-20 cells and PC12 cells are shown by immunoblotting using p84/N5 as marker for the nuclear fraction and α-tubulin as a marker for the cytoplasmic fraction. c Whole lysates were prepared from mouse brain and wild-type or Gβ5-knockout mouse pituitary gland and analyzed for Gβ₅, RGS7, or RGS9-2 by immunoblotting with the corresponding tubulin or β-actin levels shown as loading controls. d Slides of mouse pituitary were stained using control IgG, anti-Gβ₅, or anti-ACTH antibodies as indicated. The primary antibodies were visualized with Cy3-conjugated secondary antibody (red) for Gβ₅ and with FITC-conjugated secondary antibody (green) for ACTH. The slides were analyzed using confocal laser microscopy. Magnification is ×40. e Whole cell lysates from MIN6 mouse insulinoma cells or mouse brain were prepared and analyzed for Gβ₅ by immunoblotting. The corresponding β-actin levels are shown as loading controls. f Whole lysates from mouse brain or a purified preparation of pancreatic islets were prepared and analyzed for Gβ₅ by immunoblotting. The corresponding α-tubulin levels are shown as loading controls

Because AtT-20 cells were derived from a pituitary tumor, we studied Gβ₅ expression in native mouse pituitary gland. The expression of Gβ₅ as well as the Gβ₅-dependent R7-RGS proteins RGS7 and RGS9-2 was evident in wild-type, but not Gnb5 KO [3], pituitary lysate (Fig. 1c). To investigate the regional expression of Gβ₅ in mouse pituitary, we performed dual immunofluorescence staining using anti-Gβ₅ and anti-ACTH antibodies (Fig. 1d). Gβ₅ is widely expressed in both anterior and posterior pituitary lobes and it co-localizes with ACTH in the pars intermedia of the anterior murine pituitary gland (Fig. 1d).

Since Gβ₅ was expressed widely throughout both the anterior and posterior mouse pituitary we wondered whether it might also be expressed in neuroendocrine islet cells of the pancreas. Immunoblots of insulinoma-derived MIN6 mouse pancreatic islet β-cells and a purified preparation of mouse pancreatic islets demonstrated robust expression of Gβ₅ to a level comparable to that of neuronal controls (Fig. 1e, f).

Discussion

The expression of Gβ₅ and R7-RGS subfamily RGS proteins in brain and retina is well documented (reviewed in [1, 2]), yet previous study from our laboratory suggested that Gβ₅ was also expressed in cells of neuroendocrine origin, including the αT3-1 pituitary cell line [4]. In this study, besides documenting the expression of Gβ₅ in corticotroph and islet β-cell derived cell lines, we demonstrate the unequivocal expression of Gβ₅ in native endocrine tissue including cells of the pituitary and in pancreatic islet cells. The pancreatic islet cells derive from embryonic endodermal anlage, while the anterior and posterior pituitary develop from oral and neural ectoderm respectively. The expression of Gβ₅ in tissues of such disparate embryologic origins implies an important role for the Gβ₅/R7-RGS complex in neuroendocrine cells and in the physiology of endocrine systems. Indeed, targeted disruption of the Gβ₅ partner RGS7 in Rgs7^tm1Lex mice results in a phenotype of impaired glucose tolerance (Mutant Mouse Regional Resource Center, strain 011655; see http://www.mmrrc.org/strains/11655/011655.html). Furthermore, while this manuscript was being completed, Slepak and co-workers published evidence suggesting a role for Gβ₅ in the regulation of energy metabolism at the organismal level and confirming the presence of the Gβ₅ complex in cultured MIN6 cells and isolated pancreatic islets [10].

Even in the nervous system, few details of the function of the Gβ₅/R7-RGS complex at the cell biological level are understood. It is known that the Gβ₅/R7-RGS complex exhibits GTPase activating protein (GAP) activity for heterotrimeric Gα subunits and thus can function as a negative regulator of signaling to effectors from GPCRs. Studies in vitro have found that the GAP activity of Gβ₅/R7-RGS complexes is most efficacious against pertussis-sensitive G_oα subunits [11]. The expression of Gβ₅ in pituitary and islet cells makes it likely that modulation of such GPCR-coupled pertussis-sensitive pathways by the Gβ₅/R7-RGS complex may control hormone release and endocrine function in a physiologically important, and perhaps pharmacologically manipulable, fashion.