Characterization of the extra-large G protein alpha-subunit XLalphas. I. Tissue distribution and subcellular localization.
Our group previously described a new type of G protein, the 78-kDa XLalphas (extra large alphas) (Kehlenbach, R. H., Matthey, J., and Huttner, W. B. (1994) Nature 372, 804-809 and (1995) Nature 375, 253). Upon subcellular fractionation, XLalphas labeled by ADP-ribosylation with cholera toxin was previously mainly detected in the bottom fractions of a velocity sucrose gradient that contained trans-Golgi network and was differentially distributed to Galphas, which also peaked in the top fractions containing plasma membrane. Here, we investigate, using a new antibody specific for the XL domain, the tissue distribution and subcellular localization of XLalphas and novel splice variants referred to as XLN1. Upon immunoblotting and immunofluorescence analysis of various adult rat tissues, XLalphas and XLN1 were found to be enriched in neuroendocrine tissues, with a particularly high level of expression in the pituitary. By both immunofluorescence and immunogold electron microscopy, endogenous as well as transfected XLalphas and XLN1 were found to be predominantly associated with the plasma membrane, with only little immunoreactivity on internal, perinuclear membranes. Upon subcellular fractionation, immunoreactive XLalphas behaved similarly to Galphas but was differentially distributed to ADP-ribosylated XLalphas. Moreover, the bottom fractions of the velocity sucrose gradient were found to contain not only trans-Golgi network membranes but also certain subdomains of the plasma membrane, which reconciles the present with the previous observations. To further investigate the molecular basis of the association of XLalphas with the plasma membrane, chimeric proteins consisting of the XL domain or portions thereof fused to green fluorescent protein were analyzed by fluorescence and subcellular fractionation. In both neuroendocrine and non-neuroendocrine cells, a fusion protein containing the entire XL domain, in contrast to one containing only the proline-rich and cysteine-rich regions, was exclusively localized at the plasma membrane. We conclude that the physiological role of XLalphas is at the plasma membrane, where it presumably is involved in signal transduction processes characteristic of neuroendocrine cells.
Department of Neurobiology, University of Heidelberg, Im Neuenheimer Feld 364, D-69120 Heidelberg and Max-Planck-Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 110, D-01307 Dresden, Germany.
H A
HA
Pasolli
M
M
Klemke
R H
RH
Kehlenbach
Y
Y
Wang
W B
WB
Huttner
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002462:Cell Membrane / Q000737:chemistry; D005455:Fluorescent Antibody Technique; D019204:GTP-Binding Proteins / Q000032:analysis*; D006367:HeLa Cells; D006801:Humans; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D009490:Neurosecretory Systems / Q000737:chemistry; D016716:PC12 Cells; D021122:Protein Subunits; D051381:Rats
D021122:Protein Subunits; D000246:Adenosine Diphosphate Ribose; D019204:GTP-Binding Proteins