Immunoelectron microscopic identification of cytoplasmic and nuclear G _sα in S49 lymphoma cells

Abstract

The subcellular distribution of Gs alpha was characterized in S49 lymphoma cells with two polyclonal antisera directed against specific COOH- and NH2-terminal epitopes. Nonspecific binding was determined in each subcellular compartment by incubating cyc- S49 cells, known to be deficient in Gs alpha and its mRNA, with primary and secondary antisera. Small proportions of total specific binding sites were localized to the plasmalemma as well as the nuclear envelope. Because of their small size, these compartments contained a high concentration of Gs alpha. However, most of the specific binding sites were found in nonstructured cytoplasm and within the nucleus. Specific binding was abolished or significantly reduced by preincubating primary antisera with their peptide immunogens but not with an irrelevant peptide. Intracellular Gs alpha immunoreactive binding sites did not colocalize with gold-conjugated transferrin in cells preincubated with this ligand to mark a classical endocytotic pathway. The intracellular and intranuclear location of Gs alpha was confirmed with confocal microscopy of S49 cells immunostained with specific primary and fluorescently labeled secondary antibodies. Gs alpha was also detected with immunoblots of proteins extracted from purified S49 cell nuclei. Thus, Gs alpha is abundantly distributed in intracellular and intranuclear sites in S49 cells and occurs in loci distinct from organelles of the transferrin pathway. The substantial intracellular distribution of Gs alpha suggests that Gs may subserve intracellular and, perhaps, intranuclear functions that may be important in proliferating cells.

Expression of the Gs protein alpha-subunit disrupts the normal program of differentiation in cultured murine myogenic cells.

“…Proliferating C 2 C 12 myoblasts exhibited a significant fraction of total cellular G s ␣ in cytoplasmic and nuclear compartments ( Fig. 1 A ), similar to our previous findings in S49 lymphoma cells (22). In striking contrast, intranuclear G s ␣ was not detected in C 2 C 12 myotubes (Fig.…”

“…We have previously shown that a substantial portion of total cellular G s ␣ is not associated with the plasmalemma but in-stead localizes to cytoplasmic and nuclear compartments of proliferating S49 lymphoma cells (22). In contrast, we have noted similar cytoplasmic but no specific nuclear G s ␣ immunoreactivity in postmitotic-differentiated cardiac myocytes (23).…”

“…In previous works, we immunolocalized G s ␣ protein to nuclei of proliferating S49 lymphoma cells but did not detect G s ␣ in nuclei of postmitotic differentiated cardiac myocytes (22,23). To explore the significance of nuclear G s ␣ in a differentiating cell line, we immunostained proliferating C 2 C 12 myoblasts and differentiated C 2 C 12 myotubes with 3A-150, a specific antiserum raised against an epitope unique to the native G s ␣ subunit.…”

“…To explore the significance of nuclear G s ␣ in a differentiating cell line, we immunostained proliferating C 2 C 12 myoblasts and differentiated C 2 C 12 myotubes with 3A-150, a specific antiserum raised against an epitope unique to the native G s ␣ subunit. We have previously characterized this antibody and validated its use for immunocytochemical localization of G s ␣ in cells and tissues at the light and electron microscopic levels (22,23). Proliferating C 2 C 12 myoblasts exhibited a significant fraction of total cellular G s ␣ in cytoplasmic and nuclear compartments ( Fig.…”

Proinflammatory Gene Induction by Platelet-Activating Factor Mediated Via Its Cognate Nuclear Receptor

“…Separate functions for the intracellular and cell surface receptors have been suggested using agents that putatively distinguish these receptors (8,9). Specifically, immediate effects have been proposed to be mediated by cell surface receptors, whereas regulation of expression of specific genes may be dependent upon intracellular receptors, consistent with the presence of signaling effectors in nuclei, including G proteins (14,15), ionic channels (16,17), phospholipases (12,18), adenylate cyclase (19), kinases (20), and NF-B (21). However, the specific identification of intracellular PAF receptors as well as their physiological functions, especially on organelles, which may explain their presumed involvement in gene regulation (8,22) in particular nuclei, has never been explicitly demonstrated.…”