Activation of the platelet-derived growth factor beta receptor [electronic resource] : the role of the juxtamembrane cytoplasmic region.

9780599575271

1999

1 online resource (213 p.)

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The platelet-derived growth factor beta receptor (PDGFbetaR) is a transmembrane receptor tyrosine kinase (RTK) involved in cell proliferation and a variety of other cellular functions. I have generated a constitutively activated murine PDGFbetaR that contains a valine to alanine substitution at residue number 536, located in the cytoplasmic juxtamembrane (JM) domain of the receptor. When this mutant receptor (PR-V536A) was expressed in Ba/F3 cells, an IL-3-dependent murine cell line that does not express endogenous PDGFbetaR it allowed the cells to survive and proliferate in the absence of IL-3 or PDGF. Tyrosine phosphorylation of PR-V536A was markedly increased compared to that of the wild-type PDGFbetaR in the absence of ligand and similar to that observed in ligand-activated wild-type PDGFbetaR. Furthermore, a fraction of PR-V536A constitutively formed dimers at the cell surface, and the mutant receptor was constitutively bound to cellular signalling proteins. PR-V536A displayed increased tyrosine kinase activity in vitro toward an exogenous substrate, and the tyrosine kinase activity of the receptor was required for the constitutive activation of the mutant. This valine to alanine substitution also activated a PDGFbetaR mutant unable to bind PDGF. Strikingly, alanine substitutions at positions homologous to V536 of the murine PDGFbetaR also activated human alpha and beta PDGFR, colony stimulating factor 1 receptor, and stem cell factor receptor, other members of the PDGF receptor subfamily. Alanine scanning in the area surrounding residue 536 in the murine PDGFbetaR identified two other constitutively activated mutant receptors, PR-W534A and PR-I537A. The finding that several amino acid substitutions in the JM domain of PDGFbetaR cause constitutive receptor activation suggests that activation is achieved by the disruption of a function. The data presented here, together with data generated by others, strongly support the notion that the JM region of receptors of the PDGFR-subfamily of RTKs contains an inhibitory sequence that prevents receptor activation in the absence of ligand. Inspection of the amino acid sequence of this region of the receptors revealed a strong similarity to WW domains present in a number of other signal transduction proteins. Furthermore, GST fusion proteins containing the juxtamembrane region of the PDGFbetaR specifically associated with peptides containing the WW domain consensus recognition sequences PPXY and PPLP. This is the first report describing the existence of WW domains in RTKs. However, mutations at the conserved residues that define the PDGFbetaR WW domain did not cause constitutive receptor activation, nor did they prevent activation of the receptor when combined with the activating V536A substitution. Thus, neither disruption of the WW domain nor modification of its specificity appears to account for receptor activation in the JM point mutants. Interestingly, a mutant PDGFbetaR in which the two defining tryptophan residues of the WW domain were replaced by alanines displayed a reduced response to PDGF. These results suggest that the JM domain of receptors of the PDGFR-subfamily of RTKs plays several roles in the regulation of receptor activity and function, some of which may involve protein-protein interactions mediated by the WW domain. It remains to be elucidated whether the WW module and the putative inhibitory domain of these receptors exert related or independent effects on receptor function.

Books / Online / Dissertations & Theses

English

July 12, 2011

Thesis (Ph.D.)--Yale University, 1999.