The mechanisms governing the specific membrane targeting of adenylyl cyclase isoforms remain unknown. To address this issue, a series of chimeras were produced between the raft-targeted AC5 and the non-raft-targeted AC7, involving switching of their major domains. The AC5-AC7 chimeras were expressed in HEK 293 cells and lipid rafts were isolated from the bulk plasma membrane by either detergent-based or non-detergent-based fractionation methods. Additionally, confocal imaging was used to investigate the precise cellular targeting of the chimeras. Surprisingly, the two tandem six-transmembrane domains of AC5 were not required for localization to lipid rafts. Rather, AC5 localization depended on the complete cytoplasmic loops (C1 and C2); constructs with mixed domains were either retained in the endoplasmic reticulum or degraded. Similar conclusions are drawn for the lipid raft localization of the Ca 2؉ /calmodulinstimulable AC8; again, the C1 and C2 domains are critical. Thus, protein-protein interactions may be more important than protein-lipid interactions in targeting these calcium-sensitive enzymes to lipid rafts.

We have isolated a series of human testis poly(A) cDNA clones by cross-hybridization to SPGY1, a Y gene homologous to DAZ. Their sequence analysis revealed an identical nucleotide composition in different 'full-length' clones, suggesting that all were encoded by the same gene. We mapped this gene to the short arm of chromosome 3 and designated it SPGYLA (SPGY like autosomal). Comparison of the SPGYLA cDNA sequence with the cDNA sequences of DAZ and SPGY1 revealed two prominent differences. The tandem repetitive structure of 72 bp sequence units (DAZ repeats) is absent. SPGYLA contains only one 72 bp sequence unit. Downstream of it, a specific 130 bp sequence domain is present which is absent in DAZ and SPGY1 but present in the mouse gene Dazla and in the Drosophila gene boule. SPGYLA encodes an RNA binding protein expressed only in the human male gonad. The data presented give strong evidence that not DAZ but SPGYLA is the functional human homologue of Dazla and boule.

The nine membrane-bound mammalian adenylyl cyclases (ACs) contain two highly diverged membrane anchors, M1 and M2, with six transmembrane spans each and two conserved cytosolic domains which coalesce into a pseudoheterodimeric catalytic unit. Previously, the catalytic segments, bacterially expressed as soluble proteins, were characterized extensively whereas the function of the membrane domains remained unexplored. Using the catalytic C1 and C2 domains of AC type V we employed the membrane anchors from type V and VII ACs for construction of enzymes with duplicated, inverted, fully swapped and chimeric membrane anchors. Further, in the M1 membrane domain individual transmembrane spans were removed or exchanged between type V and VII ACs. The constructs were expressed in HEK293 cells, the expression levels and membrane localization was assessed by Western blotting. Cell-free basal, forskolin-, GTPgS-and G sa /GTPgS-stimulated AC activities were determined. The results demonstrate that enzymatic activities were only maintained when the M1 and M2 membrane domains were derived from either AC V or VII. Constructs with chimeric membrane domains, i.e. M1 from type V and M2 from type VII AC or vice versa, were essentially inactive although the expression levels and membrane localization appeared to be normal. The data indicate a functionally important interaction of the membrane domains of ACs in that they seem to interact in a pair-like, isoform delimited manner. This interaction directly impinges on the formation of the catalytic interface. We propose that protein±protein interactions of the AC membrane domains may constitute another, yet unexplored level of AC regulation.Keywords: adenylyl cyclase; membrane anchor; forskolin; G-protein; cAMP.Adenylyl cyclases (ACs) help to translate extracellular signals into intracellular responses. Yet, the often simultaneous expression of a panoply of AC-coupled receptors by a single cell raises the question of how does a cell solve the dilemma to discriminate between different extracellular signals when it uses in many instances primary transduction pathways that involve the same second messenger, cAMP? The cell-specific expression of distinct AC isoforms is one way to entertain parallel, yet functionally separated cAMP transduction cascades because ACs are regulated differently by various G-proteins, Ca 21 and protein kinases which directly affect the cytosolic domains [1±4]. However, the two huge membrane anchors, M1 and M2, each with six predicted transmembrane spans (TM), may represent an additional, as yet unexplored protein domain with a potential for a regulatory input. Because they are dispensable for the cyclase reaction [5,6], almost all experimental efforts to date have been directed towards characterization of the soluble catalyst, which consists of the two conserved, cytoplasmic loops, C1 and C2.Several plausible considerations would argue that the functions of M1 and M2 are not confined to serve exclusively as simple membrane anchors. First, their size of more than 3...

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