Establishment of an in vivo model facilitates B2 receptorprotein maturation and heterodimerization

Abstract

In individuals with diverse cardiovascular risk factors, signalling stimulated by the AT₁ receptor for the vasopressor angiotensin II is sensitized by heterodimerization with the receptor for the vasodepressor bradykinin, B₂. Signal sensitization and receptor heterodimerization rely on efficient maturation of the B₂ receptor protein. To assess functional features of that important cardiovascular receptor system, we established an in vivo model by using immunodeficient NOD.Scid mice for the expansion of transfected cells under physiological conditions. Compared to cultivated cells, the in vivo model strongly facilitated B₂ receptor maturation and heterodimerization. To elucidate the mechanisms underlying the enhancement of B₂ receptor protein maturation under in vivo conditions, we performed microarray gene expression profiling. Microarray analysis revealed a more than 1.7-fold up-regulation of the chaperone calreticulin upon in vivo cell expansion whereas other important members of the general chaperone system were only marginally altered. Down regulation of calreticulin expression by RNA interference confirmed the importance of calreticulin for efficient B₂ receptor maturation under in vivo conditions. Receptor proteins synthesized in the Nod.Scid cell expansion model were functionally active and sensitive to drug treatment as exemplified by treatment with the AT₁-specific antagonist losartan. Thus, we established a model system that can be used to analyze functional features of proteins in vivo by expanding transfected cells in immunodeficient NOD.Scid mice.