Reinhard Grisshammer

Functional coupling with Gαq and Gαi1 protein subunits promotes high‐affinity agonist binding to the neurotensin receptor NTS‐1 expressed in Escherichia coli

To analyze the coupling of Gα subunits to the rat neurotensin receptor NTS‐1 (NTR), fusion proteins were expressed in Escherichia coli with various Gα subunits covalently linked to the receptor C‐terminus. The presence of Gαq or Gαi/q, in which the six C‐terminal residues of Gαi1 were replaced with those from Gαq, increased the percentage of receptors in the agonist high‐affinity state. This effect was less pronounced for wild‐type Gαi1 and not observed for Gαi/s. Functional coupling of neurotensin receptor to Gα was demonstrated by neurotensin‐induced [35S]GTPγS binding for the Gαq, Gαi/q and Gαi1 subunits, but not for Gαi/s. Our results extend previous findings of the dual coupling of NTR to pertussis toxin‐sensitive and ‐insensitive G‐proteins in Chinese hamster ovary cells with preference for the latter.

Expression in Escherichia coli and characterisation of the human central CB1 and peripheral CB2 cannabinoid receptors

The human central CB1 and peripheral CB2 cannabinoid receptors were expressed in Escherichia coli as fusion proteins with the maltose-binding protein at their amino-termini and a hexa-histidine/Flag tag at their carboxyl-termini. Western blot analysis of the expressed proteins revealed considerable degradation of the CB1 fusion, which failed to bind either the cannabinoid agonist CP 55,940 or the CB1-specific antagonist SR 141716A. In contrast, the CB2 fusion was well-expressed and bound several cannabinoids with affinities comparable to those observed in mammalian expression systems.