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Mutational analysis of the R33-encoded G protein-coupled receptor of rat cytomegalovirus: identification of amino acid residues critical for cellular localization and ligand-independent signalling

¹ Department of Medical Microbiology, Cardiovascular Research Institute Maastricht, University of Maastricht, PO Box 5800, 6202 AZ Maastricht, The Netherlands
² Division of Medicinal Chemistry, Leiden/Amsterdam Centre for Drug Research, Free University, 1081 HV Amsterdam, The Netherlands

Correspondence
Cornelis Vink
kvi{at}lmib.azm.nl

The rat cytomegalovirus (RCMV) R33 gene encodes a G protein-coupled receptor (GPCR), pR33, which possesses agonist-independent, constitutive signalling activity. To characterize this activity further, we generated a series of point and deletion mutants of pR33. Both expression of and signalling by the mutants was evaluated. Several point mutants were generated that contained modifications in the NRY motif. This motif, at aa 130–132 of pR33, is the counterpart of the common DRY motif of GPCRs, which is known to be involved in G protein coupling. We found that mutation of the asparagine residue within the NRY motif of pR33 (N¹³⁰) to aspartic acid resulted in a mutant (N¹³⁰D) with similar signalling characteristics to the wild-type (WT) protein, indicating that N¹³⁰ is not the determinant of constitutive activity of pR33. Interestingly, a mutant carrying an alanine at aa 130 (N¹³⁰A) was severely impaired in G_q/11-mediated, constitutive activation of phospholipase C, whereas it displayed similar levels of activity to pR33 in G_i/0-mediated signalling. Another protein that contained a modified NRY motif, R¹³¹A, did not show constitutive activity, whereas mutants Y¹³²F and Y¹³²A displayed similar activities to the WT receptor. This indicated that residue R¹³¹ is critical for pR33 function in vitro, whereas Y¹³² is not. Finally, we identified two consecutive arginines within the C-terminal tails of both pR33 and its homologue from human CMV, pUL33, which are important for correct cell-surface expression of these receptors.

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