SITE-DIRECTED MUTAGENESIS OF THE HUMAN BETA(3)-ADRENOCEPTOR - TRANSMEMBRANE RESIDUES INVOLVED IN LIGAND-BINDING AND SIGNAL-TRANSDUCTION

All three subtype of beta-adrenoceptors are coupled to stimulation of adenylyl cyclase activity via the stimulatory guanine-nucleotide-bindi ng protein. Nevertheless, the beta(3) adrenoceptor (beta(3)-AR) differ s significantly from the other subtypes in terms of pharmacology. Most strikingly, it recognizes as agonists several compounds acting as pot ent beta(1)-AR and beta(2)-AR antagonists Furthermore, the human beta( 3)-AR is quite different from the animal beta(3)-AR. Molecular modelli ng studies followed by site-directed mutagenesis tvas used here to ide ntify some of the amino acid residues which may be implicated in ligan d binding and signal transduction of the beta(3)-AR. Three contiguous residues, valine-leucine-alanine, which are present in the first trans membrane domain at positions 48-50 of the human receptor brit are abse nt in all known rodent sequences. were thought to be important for spe cies specificity. When these three residues were deleted from the huma n receptor, no 'rodent-like` pharmacological profile was obtained in t erms of either binding or adenylyl cyclase activation. Glycine at posi tion 53, also in the first transmembrane domain in the human beta(3)-A R has been suggested to participate in beta(2)-1 beta(3)-AR subtype se lectivity. Replacement of this glycine residue by phenylalanine, which is the residue present at the homologous position in the human beta(2 )-AR, left the beta(3)-AR pharmacological profile unaltered in terms o f specificity and selectivity. Aspartate residue 117, in the third tra nsmembrane domain, has been found to be essential for ligand binding a nd consequently adenylyl cyclase activation in several bioamine recept ors. When this residue was replaced by a leucine residue in the beta(3 )-AR. ligand binding and signal transduction were suppressed. Finally, replacement of asparagine at position 312 in the sixth transmembrane domain by an alanine residue. led to alterations in the signal-transdu ction pathway. pathway.