Homologous domains embedded in multi-domain proteins of different domain architectures may exhibit subtle, but important, differences in their structure and function. Here we consider two multi-domain proteins, ARNO and GRK2, which have very different domain architectures but both contain pleckstrin homology (PH) domains. We analyzed the roles of residues selectively conserved in these sub-families of PH-domains from ARNO and GRK2 proteins. Domain architecture-specific residues in PH-domain are found to contribute to structural and functional specialization of ARNO and GRK2 in terms of (i) specific intra- and inter-protein interactions, (ii) specificity for phospholipids and (iii) participation in conformational excursions, leading to various functional forms. Our approach can also be applied to subfamilies of other protein families to identify subfamily-specific residues and their specialized roles.