Amino- and carboxy-terminal deletion mutants of gsα are localized to the particulate fraction of transfected COS cells

Yong Sung Juhnn, Teresa L.Z. Jones, Allen M. Spiegel

Research output: Contribution to journalArticle

14 Scopus citations

Abstract

To elucidate the structural basis for membrane attachment of the α subunit of the stimulatory G protein (Gsα), mutant Gsα cDNAs with deletions of amino acid residues in the amino and/or carboxy termini were transiently expressed in COS-7 cells. The particulate and soluble fractions prepared from these cells were analyzed by immunoblot using peptide specific antibodies to monitor distribution of the expressed proteins. Transfection of mutant forms of Gsa with either 26 amino terminal residues deleted (Δ3-28) or with 59 amino terminal residues deleted (Δ1-59) resulted in immunoreactive proteins which localized primarily to the particulate fraction. Similarly, mutants with 10 (Δ385-394), 32 (Δ353-384), or 42 (Δ353-394) amino acid residues deleted from the carboxy terminus also localized to the particulate fraction, as did a mutant form of Gsα lacking amino acid residues at both the amino and carboxy termini (Δ3-28)/(Δ353-384). Mutant and wild type forms of Gsα demonstrated a similar degree of tightness in their binding to membranes as demonstrated by treatment with 2.5 M NaCl or 6 M urea, but some mutant forms were relatively resistant compared with wild type Gsα to solubilization by 15 mM NaOH or 1% sodium cholate. We conclude that: (a) deletion of significant portions of the amino and/or carboxyl terminus of Gsα is still compatible with protein expression; (b) deletion of these regions is insufficient to cause cytosolic localization of the expressed protein. The basis of Gsα membrane targeting remains to be elucidated.

Original languageEnglish (US)
Pages (from-to)523-530
Number of pages8
JournalJournal of Cell Biology
Volume119
Issue number3
DOIs
StatePublished - Nov 1992

ASJC Scopus subject areas

  • Cell Biology

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