Intracellular retention of membrane-anchored v-sis protein abrogates autocrine signal transduction.

Abstract

Abstract. An important question regarding autocrine transformation by v-sis is whether intracellularly activated PDGF receptors are sufficient to transform cells or whether activated receptor-ligand complexes are required at the cell surface. We have addressed this question by inhibiting cell surface transport of a membraneanchored v-sis protein utilizing the ER retention signal of the adenoviral transmembrane protein E3/19K. A v-sis fusion protein containing this signal was retained within the cell and not transported to the cell surface as confirmed by immunofluorescent localization experiments. Also, proteolytic maturation of this protein was suppressed, indicating inefficient transport to postGolgi compartments of the secretory pathway. When compared with v-sis proteins lacking a functional retention signal, the ER-retained protein showed a diminished ability to transform NIH 3T3 cells, as measured by the number and size of foci formed. In newly established cell lines, the ER-retained protein did not downregulate PDGF receptors. However, continued passage of these cells selected for a fully transformed phenotype exhibiting downregulated PDGF receptors and proteolytically processed v-sis protein. These results indicate that productive autocrine interactions occur in a post-ER compartment of the secretory pathway. Transport of v-sis protein beyond the Golgi correlated with acquisition of the transformed phenotype. Furthermore, suramin treatment reversed transformation and upregulated the expression of cell surface PDGF receptors, suggesting an important role for receptor-ligand complexes localized to the cell surface.

Transfer of endoplasmic reticulum and Golgi retention signals to human immunodeficiency virus type 1 gp160 inhibits intracellular transport and proteolytic processing of viral glycoprotein but does not influence the cellular site of virus particle budding.

“…The retention signals employed to generate Env-TM\Golgi and Env856ER with altered subcellular localizations have previously been demonstrated to confer localization to the cis-Golgi complex and ER, respectively (Swift & Machamer, 1991 ;Jackson et al, 1990Jackson et al, , 1993Lee & Donaghue, 1992), and the evidence presented in this paper supports the view that this is also the case here.…”

“…The heterologous nucleotide sequence was flanked 5h and 3h with complementary env sequences. The amino acid sequence reported to mediate residency in the ER, DEKKMP (Jackson et al, 1990(Jackson et al, , 1993Lee & Donaghue, 1992), followed by a translational stop codon, was generated at the C terminus of gp160 by inserting the appropriate nucleotide sequence 3h of nucleotide 8821 (to yield Env856ER). The 21 nucleotides encoding the six inserted amino acids plus the stop codon were flanked 5h and 3h with complementary env sequences.…”

“…membrane anchor of gp160, located between amino acids 684 and 705, has been replaced by the first membrane-anchor domain of the mouse infectious bronchitis virus M protein (Swift & Machamer, 1991), which has been reported to mediate residency within the Golgi compartment. The sequence DEKKMP, derived from the C terminus of the adenovirus E3\19K protein and reported to mediate localization to the ER (Nilsson et al, 1989 ;Jackson et al, 1990Jackson et al, , 1993Lee & Donaghue, 1992), is present at the C terminus of Env856ER.…”

The Nature of Intracrine Peptide Hormone Action

“…[1][2][3][4][5][6][7][8][9][10][11] Subsequently, evidence supporting intracrine action has been developed for a large number of peptide hormones and factors (for example, insulin, growth hormone, prolactin, nerve growth factor, interferon-␥, fibroblast growth factor [FGF], Tat protein, platelet-derived growth factor [PDGF], epidermal growth factor [EGF], parathyroid hormone-related protein [PTHrP], endogenous opiates, angiogenin, among others). [12][13][14][15][16][17][18][19][20][21][22][23][24] Although the intracellular actions of steroid hormones and their receptors have been studied intensively, the implications, if any, of intracrine peptide hormone action for normal or abnormal cellular physiology remain unclear. 23 Indeed, the default view appears to be that if such intracellular binding/action of peptide hormones exists at all, it represents a vestigial or unimportant aspect of biology.…”

Analysis of endoproteolytic cleavage and intracellular transport of human immunodeficiency virus type 1 envelope glycoproteins using mutant CD4 molecules bearing the transmembrane endoplasmic reticulum retention signal

“…Furthermore, it has been shown that the luminal (KDEL) and transmembrane ER retention (KKXX or XKXX) signals were sufficient and necessary to retain heterologous soluble and membrane-anchored proteins, respectively, in the ER (Nilsson et al, 1989;Munro & Pelham, 1987;Buonocore & Rose, 1990;Shin et al, 1991 a;Lee & Donoghue, 1992). Recently, Buonocore & Rose (1990 have demonstrated that the expression of soluble CD4 (sCD4) KDEL in CD4 + T cells or HeLa blocked the transport of gp120/gp41 complexes to the cell surface, and the T cells expressing sCD4~KDEL could not produce infectious virus particles, thereby preventing virus spread in HIV-infected cultures.…”