Alanine Scanning Mutagenesis Study of Transmembrane Helix 3 in the Ml Muscarinic Receptor

Transmeinbrane helix 3 (TM3) in the ml muscarinic receptor contains a n urn her of s t rough conserved ami no acids. Several of these residues have defined functions. 'I he functions of other conserved residues are unknown. We have used alanine canning mutagenesis to probe the functions of all of t lie rendue-' between Set 1 12 and Phel2I of the ml mACIiR. These ainino acids fall into four basic clauses and appear to be organised into coherent sectors on the siirfaie of TXU The first contains Mll-i. L118 and F121. where there was little efieri on binding or signalling. These residues are modelled to face away from the core of the p-crptor stnic.turo, towards the phospholipid bilayer. The seu>ml consi.-ts ot M 1") arid LI 17. which resemble 1)122 and VI2-1 in thai ilieir muta lion rnainlv affects receptor expression level, but not binding or signalling. In a helical projec t ion model, they lie almost directly above 1)122 or Y12 1. faring toward TM2 01 TM4/5. The third contains VlKf. LtHi and S120. in tinj. <ase. mutation increases A('h affinity, and potency in mediating the phospho inositide reMponse. These residues may. together, participate in intramoleculai constraint-4 on the agonist-indured conformational change, and may also haAf iinportance for receptor expression levels. The fourth comprises SI 12 and li 1'). Muiation of the-4e residues, like 1I123, strongly inhibits signalling to i he phos phomosiiide pathway, while having smaller effects on binding, and little effect on re( eplot expression. '1 hese amino acids are oriented towards TM7. and max participate in maintaining a conformation of the receptor which i.- necesar> for (i pruti-ifi -4linding. Snpiport: MHC and Wellcome Trust.