CRYPTIC INITIATION AT THE HUMAN D4 RECEPTOR REVEALS A FUNCTIONAL-ROLEFOR THE AMINO-TERMINUS

It was found that deletion of the initiator methionine of the D4 recep tor results in the use of a cryptic initiation site in the putative fi rst transmembrane region. We made use of this observation to investiga te the role of the amino terminus of the D4 receptor. In vitro transcr iption and translation of D4.4 and a D4.4 deleted for the initiation c odon (D4.4 Delta NH2) resulted in the formation of protein products wi th a molecular mass of about 44 and 40.5 kDa, respectively. The molecu lar mass of 40.5 kDa suggests initiation in the putative first transme mbrane region. Transient expression of various deletion mutants indica ted that this receptor form can be expressed at up to 70% of the D4.4 control levels and provided support for the existence for an alternati ve translation initiation site in the first transmembrane domain, most likely at nucleotide +112 (the initiator methionine codon is designat ed as +1). The D4.4 Delta NH2 mutant was stably expressed in CHO cells . Pharmacological analysis demonstrated no major differences in antago nist binding with the regular D4.4 receptor, while dopamine and quinpi role binding affinities were about 5-fold decreased. The half-maximal level (EC(50)) for blocking forskolin-stimulated cAMP levels by dopami ne was about 10-fold lower as compared to D4.4, Furthermore, the funct ional efficacy is decreased by about 40%. These data suggest that the amino-terminal domain is not essential for proper expression, but does interfere with the functional activity of the receptor, possibly thro ugh stabilization of the active state. To our knowledge this is the fi rst demonstration that the amino terminus of a dopamine receptor is in volved in signal transduction.